Analysis of SCAR marker nucleotide sequences in maize (Zea mays L.) somaclones.

SCAR (sequence characterized amplified region) markers allow the reliable identification of unique somaclonal variations. Six SCAR markers were developed previously and were thought to be exclusively characteristic of eight maize somaclones. However, we detected two of these markers in maize lines and a cultivar unrelated to the progenitor line of the somaclones. Therefore, we sequenced these markers and performed bioinformatic searches to understand the molecular events that may underlie the variability observed in the somaclones. All changes were found in noncoding sequences and were induced by different molecular events, such as the insertion of long terminal repeat (LTR) transposon(s), precise miniature inverted repeat transposable element (MITE) excision, microdeletion, recombination, and a change in the pool of mitochondrial DNA. For example, the SCAR marker QR is represented by the two variants QR-A and QR-2. The sequences of the two variants were similar, except for a 457-bp fragment found only in QR-A; this region was denoted as Q. Region Q was flanked by the direct 3-bp repeat 5'-TAA-3' (target site duplication; TSD) and the inverted 14-bp repeat 5'-GGGCCTGTTTGGAA-3' (terminal inverted repeats; TIRs). These features confer the Q region with similarity to the nonautonomic Tourist-like MITE. In two groups of independently produced somaclones, the same features (morphological, molecular) were variable, which confirms the theory of 'hot spots' occurring in the genome. The distribution of one of the SCAR markers was confirmed using Southern blot hybridization. The presence of the same molecular markers in the somaclones and in different non-somaclonal maize variants suggests that in some cases, the same mechanisms determine both in vitro and in vivo variability and that cell culture enhances the rate of heritable genomic changes that naturally occur in living organisms.

[New allele of the COCHLEATA gene in pea Pisum sativum L].

Analysis with the polymerase chain reaction showed that the Khlorofill-4 pea Pisum sativum chlorophyll-deficient mutant with reduced stipules has an altered structure of the COCHLEATA (COCH) gene, carrying a new mutant COCH allele. The phenotype of the mutant was described in comparison with another form having reduced stipules (stipules reduced) and the control. Leaves of the coch mutant are smaller and have other proportions than in the control; stipules are absent from leaves of the first nodes and are narrow, bandlike, or spoonlike at later ontogenetic stages. It was concluded that the cell number in the stipule epidermis is reduced in the st and coch mutants compared to the wild type.

[Allele polymorphism of microsatellite loci in pea Pisum sativum L. lines, varieties, and mutants].

Interlinear polymorphism at 23 microsatellite loci was studied in 40 Pisum sativum lines, varieties, and mutants and proved to be high, 61.6% on average. Varieties bred for different end uses substantially differed in the extent of polymorphism and allele composition. Polymorphism of microsatellite loci was shown to be suitable for developing passports of industrial pea varieties.

[CAPS markers for the identification of garden pea (Pisum sativum L.) cultivars].

The CAPS (PCR-PDRF) method was used to analyze polymorphism in sequences of unique genes among specimens of 24 pea lines and cultivars. Analysis of each employed molecular-genetic marker was found to reveal three to seven polymorphic sequence variants. Analysis with the use of five selected markers allows the unambiguous identification of any of examined specimens. Thus, the possibility of using CAPS markers for identification and classification of garden pea cultivars has been shown. Possible prospects for this approach and the ways of its further implementation are considered.

[Genetic control of fasciation in pea (Pisum sativum L.)].

The inheritance and manifestation of fasciation character in three fasciated lines of common pea Pisum sativum L. were investigated. All studied forms are characterized by abnormal enlargement of stem apical meristem leading to distortions in shoot structure. It was estimated that fasciation in mutant Shtambovyi is connected with recessive mutation in gene FAS, which was localized in linkage group III using morphological and molecular markers. It was demonstrated that fasciation in cultivar Rosacrone and line Lupinoid is caused by recessive mutation of the same gene (FA). The peculiar architecture of inflorescence in the Lupinoid line is a result of interaction of two recessive mutations (det fa). Investigation of interaction of mutations fa and fas revealed that genes FA and FAS control consequential stages of apical meristem specialization. Data on incomplete penetrance and varying expressivity were confirmed for the mutant allele fa studied.

[The study of the genetic effects in generation of pea plants cultivated during the whole cycle of ontogenesis on the board of RS ISS].

Results of studies on growth and development of offspring of two genetically marked dwarf pea lines planted during the whole ontogenesis cycle in the Lada space greenhouse on board of Russian Segment of International Space Station (RS ISS) are presented. The offspring of M1 and M2 plants grown from seeds formed during space flight was examined under conditions of Earth-based. Cultivation. It had been shown that growth and developmental characteristics, frequency of chromosome aberrations in primary root meristem and level of molecular polymorphism revealed in individual plants via RAPD method show no significant differences between offspring of "space-grown" and control seeds.

[Analysis of DNA polymorphism in a relict Uralian species, yellow foxglove (Digitalis grandiflora Mill.), using RAPD and ISSR markers].

Genetic polymorphism of the Uralian relict plant species, yellow foxglove Digitalis grandiflora Mill. (family Scrophulariaceae), was examined using RAPD and ISSR techniques. A total of 149 RAPD and 74 ISSR markers were tested. The indices characterizing polymorphism and genetic diversity were calculated. The data obtained pointed to a high level of genetic variation of D. grandiflora (P95 = 65%). The cenopopulation examined was weakly differentiated with most of genetic diversity accounted by within-population differentiation.

[Fasciation in pea: basic principles of morphogenesis].

A study of fasciated pea Pisum sativum L. (Fabaceae) mutant Shtambovy in comparison with the wild type (Nemchinovsky cultivar) has shown that fasciation is a result of abnormal cohesion of axial or other structures which arise in a superfluous amount due to uncontrolled meristic processes. In some cases, the organs with the same number and position as in the wild type can be fascinated. Subsequent defasciation and some features of tissue differentiation suggest that the meristem of a fasciated shoot retains a certain degree of discreteness which reflects its complex structure. The number and position of leaves in a node is a function of the diameter of the leaf primordium inhibitory zone, size of the shoot apical meristem, and number of bundles in a shoot. In the absence of the apex proliferative activity combined with the reduction of phyllomes in the upper nodes, abnormal cohesion of the second order axes, racemes, can take place. As a result, inflorescences of special type develop.

[Development and study of SCAR markers in pea (Pisum sativum L.)].

In order to develop more specific markers that characterize particular regions of the pea genome, the data on nucleotide sequences of RAPD fragments were used for choosing more extended primers, which may be helpful in amplifying a fragment corresponding to the particular DNA region. Of the 14 STS markers obtained from 14 polymorphic RAPD fragments, 12 were polymorphic, i.e., they are SCAR markers that can be used in genetic analysis. The transition from complex RAPD spectra to amplification of a particular SCAR marker substantially facilitates analysis of large samples for the presence or absence of the examined fragment. Inheritance of the developed SCAR markers was studied in F1 and F2. SCAR markers were used to identify various pea lines, cultivars, and mutants. It was established that the study of amplification of STS markers in various pea genotypes at varying temperatures of annealing and the comparison with amplification of the original RAPD fragments in the same genotypes provide an approach for analysis of RAPD polymorphism type.

[Studying plant genome variation using molecular markers].

The authors' studies on the organization and variation of plant genome with the use of molecular markers are briefly reviewed with special emphasis on random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), sequence characterized amplified region (SCAR), and cleaved amplified polymorphic sequence (CAPS) markers detected with the use of polymerase chain reaction (PCR). These markers have been demonstrated to be promising for identifying cultivars and determining the purity of genetic strains of pea. Genetic relationships between strains, cultivars, and mutants of pea have been studied. The role of molecular markers in molecular genetic mapping and localizing the genes of commercially important characters of pea has been shown. The possibility of the use of molecular markers for studying somaclonal variation and detecting mutagenic factors in plants during long-term spaceflights is considered. The prospects of using DNA markers for understanding the organization and variability of higher plant genomes are discussed.

[RAPD and ISSR analyses of regenerated pea Pisum sativum L. plants].

Long-term pea callus cultures of different genotypes (mutants R-9 and W-1 and cultivar Viola) were used to regenerate plants (generation R0). The regenerants displayed changes both in qualitative and in quantitative traits. The most dramatic morphological alterations and complete sterility were observed in regenerants of the cultivar Viola. To estimate the genetic differences, regenerants were compared with the original lines with the use of RAPD (random amplified polymorphic DNA) and ISSR (inter simple sequence repeat) analyses. The extent of divergence varied among regenerants and depended mostly on the original genotype. The genetic difference from the original line was no more than 1% in W-1 regenerants, 0.7-5.3% in R-9 regenerants, and 10-15% in sterile regenerants of the cultivar Viola. The genetic variation of plants regenerated from a callus culture maintained for ten years did not exceed that of plants obtained from a culture maintained for two years.

[Growth, development and genetic status of pea plants cultivated in space greenhouse "LADA"].

In the period between March 2003 and April 2005 five crops of genetically marked dwarf pea were cultivated in greenhouse LADA on the ISS Russian segment to study morphology and genetics in consecutive generations. Results of the first space experiment were analyzed and characteristics of ground plants grown from space seeds were studied. It was shown that parameters of growth, development and genetic status of pea plants that completed the ontogenetic cycle in LADA did not differ substantially from the laboratory control and that plants cultivated in space flight do not incur losses in the reproductive function and form viable seeds. Genetic analysis of plants from the first generation of space and ground seeds using the methods of RAPD-primers (10 markers) and chromosomal aberration analysis failed to reveal genetic polymorphism which means that the spaceflight factors had no effect on the genetic apparatus of the first generation of space-grown plants.

[Identification and mapping of chi115 gene and DNA markers linked to it in pea (Pisum sativum L.)]. / Identifikatsii i lokalizatsii gena chi115 i stseplennykh stseplennykh s nim DNK-markerov u gorokha posevnogo (Pisum sativum L.)

Chlorophyll mutant Chi115 was induced by ethylmethane sulfonate (EMS) treatment of seeds of genotype Torsdag in Moscow State University and is characterized by lighter plant color. The monogenic nature of the mutant was determined by analyzing the F2 population from a cross between two P. sativum genotypes, WL1238 and Chi115. To establish a local map around the chi115 gene, the RAPD and ISSR techniques were used with 45 RAPD and 10 ISSR primers in combination with bulked segregant analysis (BSA). Linkage of 12 RAPDs and 2 ISSRs to the chi115 locus was observed in analysis of F2 single plants. Two RAPD markers that were closely associated with the chi115 gene were converted into the sequence characterized amplified region (SCAR) markers. By lowering the LOD score to 2, the linkage group containing the chi115 gene could be linked to the b gene (color of the flower) on linkage group III. Nevertheless, to prove the result obtained, three CAPS markers Sodmt, TubA1, and Rb were chosen on linkage group III. The results of linkage analysis showed that these CAPS markers were located within the linkage group including the chi115 gene.

[Analysis of specific RAPD- and ISSR-fragments in somaclonal maize (Zea mays L.) and development of SCAR markers based on them]. / Vyiavlenie spetsificheskikh RAPD- i ISSR-fragmentov u somaklonov kukuruzy (Zea mays L.) i sozdanie na ikh osnove SCAR-markerov.

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats) markers were used to analyse the genetic divergence between the regenerated plants derived from callus cultures and the original maize line A188. Analysis of polymorphism by using 38 RAPD- and 10 ISSR-oligonucleotide primers showed that the differences between eight examined somaclones and the original line ranged from 6.5 to 23%. As confirmed using new primers, the regenerants derived from callus cultures grouped into two clusters according to their origin. The regenerants isolated from calluses grown for eight months differed from one another and the original line to a larger extent than the regenerants obtained from two-month callus cultures. In some somaclones, molecular marking of the regenerants revealed specific RAPD and ISSR fragments that were absent in other somaclones or the original maize line. On the basis of six specific fragments (five RAPD and one ISSR), SCAR (Sequence Characterized Amplified Region) markers were developed. Specific polymorphism revealed with random primers was completely confirmed using five SCAR markers. Polymorphism of one SCAR marker differed from that revealed with random primers. Five SCAR fragments were inherited as simple dominant traits. One SCAR fragment displayed codominant inheritance.

[Production of a SCAR marker in pea Pisum sativum L. using RAPD analysis]. / Sozdanie SCAR-markera u gorokha (Pisum sativum L.) na osnovanii RAPD-analiza.

A polymorphic 750-bp fragment, RAPD marker, specific to particular pea genotypes (line L-111 and the Nord cultivar) was identified. Using this RAPD marker, SCAR was obtained. SCAR inheritance in the first and second generations was studied and its dominant character was shown.

[RAPD-analysis of corn somaclones]. / RAPD-analiz somaklonov kukuruzy.

The genetic difference between maize line A188 and A188-derived somaclones was assessed via analysis of randomly amplified polymorphic DNA (RAPD). In total, 15 out of 17 decanucleotide primers used each allowed amplification of 2-17 fragments ranging 200-2000 bp. The RAPD patterns did not differ between individual plants of line A188, which demonstrated again its high genetic homogeneity. The difference between the initial line and the somaclones was high, ranging 64-74%. On evidence of the genetic divergence, the somaclones formed two clusters. The distribution of somaclones between these clusters was consistent with their origin.

[Photochemical activity of pea mutants with damaged photosystems]. / Issledovanie fotokhimicheskoi aktivnosti mutantov gorokha s narusheniem fotosistem.

The interrelationship between biochemical photosynthetic reactions and mutations was investigated, using five nuclear recessive lethal pea mutants with damaged photosystems II (1, 19) and I (5, 21, 22). Based on the data from photoreduction of NADP+, light-induced redox conversions of exogenous and endogenous plastocyanines in isolated chloroplasts, light-induced redox conversions of cytochrome f and absorbance changes at 520 nm in chloroplasts and leaves, the possible existence of alternative pathways of electron transfer and the pleiotropic effects of mutations, are discussed. A correlation between the structural damages of chloroplasts and the photochemical activity of mutants was found. The existence of cyclic electron transport around photosystem I involving cytochrome f and plastocyanine in mutants 1 and 19 and of an electron transfer pathway from H2O to NADP+ without plastocyanine in mutant 22 is postulated.

[Some peculiarities of structure and protein composition of chloroplast membranes of pea mutant chlorophyll I]. / Osobennosti stroeniia i belkovogo sostava membran khloroplastov mutanta gorokha khlorofill 1.

The morphological properties and polypeptide composition of inner chloroplast membranes of normal and mutant pea plants (nuclear mutant "Chlorophyll I") were compared. The total fractions of chloroplast membranes from both sources were separated in a stepwise sucrose density gradient resulting in two fractions. The major fraction from mutant plants, unlike that from normal plants, does not contain thylakoids of the grana but is represented by reticular and cellulate structures, in which each cell corresponds to a thylakoid of macrograna and whose size exceeds that of normal 3 - 4-fold. In addition the inner membranes of mutant plant chloroplasts do not contain membrane polypeptides with molecular weight of 32 000 and 35 000.