Co-dominant SCAR Markers Research Articles

Deciphering gene specific molecular characterization and partial gene sequence for combined resistance to tomato leaf curl virus (ToLCV) (Ty-1, Ty-2 and Ty-3), fusarium wilt (I-2) and root- knot nematode (Mi-1) in selected fresh market breeding line of tomato (Solanum lycopersicom L.).

Tomato (Solanum lycopersicum L.) is a widely cultivated crop in tropical regions, but its production is often hampered by significant losses attributed to diseases like tomato leaf curl virus (ToLCV), fusarium wilt and root-knot nematode. This study employed an integrated approach utilizing both co-dominant and dominant SCAR markers, selected for specific resistance genes (ToLCV-Ty-1, Ty-2, Ty-2, Fusarium wilt (Race-2)-I-2, and Root-knot nematode-Mi-1. These markers with their specific gene of interest were used to screen the ten fresh market breeding lines of tomato. The P625 marker played a pivotal role in the identification process of Ty-3 alleles and effectively distinguishing between Ty-3a and Ty-3. I-2/5 (Fusarium wilt I-2), and Mi-23 (Root-knot nematode Mi-1) effectively identified and discriminated between heterozygous and homozygous states of specific genes. All resistant lines and a susceptible line for ToLCV (Ty-1, Ty-2, Ty-3), Fusarium wilt ((Race-2)- I-2) and Root-knot nematode underwent sequencing using specific primer pairs through the Sanger dideoxy technique. The resulting nucleotide sequences were aligned utilizing MEGA7 bioinformatic software and subjected to nucleotide BLAST in the NCBI database to determine sequence per cent identity and query cover, facilitating comparison with other submitted sequences. The determination of genomic positions for these nucleotide sequences may enable researchers to cartographically pinpoint the locations of genetic variations within the genome.

ОЦЕНКА АЛЛЕЛЬНОГО СОСТОЯНИЯ ГЕНА FAOMT У СЕЛЕКЦИОННЫХ ФОРМ ЗЕМЛЯНИКИ МЕЖСОРТОВОГО ПРОИСХОЖДЕНИЯ

Improving the fruit aroma is one of the promising directions of modern strawberry breeding. The strawberry fruit aroma is a complex trait, formed by a combination of a large number of volatile compounds. The present study shows the results of assessing the allelic state of the FaOMT gene, determining biosynthesis of mesifurane (volatile compound) in strawberry selected forms created in the «I.V. Michurin FSC». The biological objects of the study were 22 strawberry selected intervarietal forms from 12 crossing combinations. The allelic state of the FaOMT gene was identified using codominant SCAR marker FaOMT-SI/NO. As a result of the studies, the marker fragment of the functional allele FaOMT+ was identified in 86.4 % of forms. A heterozygous combination of alleles of the FaOMT gene (average level of mesifurane accumulation in fruits) was detected in 9.1 % forms. The homozygous state of the functional allele was detected in 77.3% forms. The homozygous state of the non-functional allele FaOMT- (mesifurane is not produced) is characterized by three genotypes (13.6 %). The greatest value for strawberry breeding use are forms with the homozygous state of the FaOMT+ allele: 1/6-41 (Vima Zanta × Polka), 2/2-32, 2/2-41 (Faith × Tea), 3/2-62 (Vima Zanta × Privlekatelnaya), 3/4-6, 3/4-7 (Malwina × Tea), 3/9-5, 3/9-12 (Florence × Faith), 5/1-105 (Polka × Vima Zanta), 5/2-23, 5/2-26, 5/2-32 (San Andreas × Monterey), 6/3-5, 6/3-21 (Kimberly × 9/2-2), 7/2-78 (Asia × Maya), 9/2-2, 9/2-7 (Kimberly × Honeoye). These forms are characterized by the maximum level of mesifurane biosynthesis in fruits and their involvement of which in hybridization allows the transmission of the target allele to 100 % of the hybrid seedlings.

Development of co-dominant SCAR markers linked to resistant gene against the Fusarium oxysporum f. sp. radicis-lycopersici.

We developed highly reliable co-dominant SCAR markers linked to the Frl gene. FORL testing is difficult. The marker is expected to be quickly adapted for MAS by tomato breeders. Fusarium oxysporum f. sp. radicis-lycopersici causes Fusarium crown and root rot (FCR), an economically important soil-borne disease of tomato. The resistance against FCR is conferred by a single dominant gene (Frl) located on chromosome 9. The aim of this study was to develop molecular markers linked to the Frl gene for use in marker-assisted breeding (MAS) programs. The FCR-resistant 'Fla. 7781' and susceptible 'B560' lines were crossed, and F1 was both selfed and backcrossed to 'B560' to generate segregating F2 and BC1 populations. The two conserved set II (COSII) markers were found linked to the Frl gene, one co-segregated with FCR resistance in both F2 and BC1 populations and the other was 8.5 cM away. Both COSII markers were converted into co-dominant SCAR markers. SCARFrl marker produced a 950 and a 1000 bp fragments for resistant and susceptible alleles, respectively. The linkage of SCARFrl marker was confirmed in BC2F3 populations developed by backcrossing the resistant 'Fla. 7781' to five different susceptible lines. The SCARFrl marker has been in use in the tomato breeding programs in BATEM, Antalya, Turkey, since 2012 and has proved highly reliable. The SCARFrl marker is expected to aid in the development of FCR-resistant lines via marker-assisted selection (MAS).

Development of IRAP- and REMAP-derived SCAR markers for marker-assisted selection of the stripe rust resistance gene Yr15 derived from wild emmer wheat.

Yr15 provides broad resistance to stripe rust, an important wheat disease. REMAP- and IRAP-derived co-dominant SCAR markers were developed and localize Yr15 to a 1.2 cM interval. They are reliable across many cultivars. Stripe rust [Pucinia striiformis f.sp. tritici (Pst)] is one of the most important fungal diseases of wheat, found on all continents and in over 60 countries. Wild emmer wheat (Triticum dicoccoides), which is the tetraploid progenitor of durum wheat, is a valuable source of novel stripe rust resistance genes for wheat breeding. T. dicoccoides accession G25 carries Yr15 on chromosome 1BS. Yr15 confers resistance to virtually all tested Pst isolates; it is effective in durum and bread wheat introgressions and their derivatives. Retrotransposons generate polymorphic insertions, which can be scored as Mendelian markers using techniques such as REMAP and IRAP. Six REMAP- and IRAP-derived SCAR markers were mapped using 1,256 F2 plants derived from crosses of the susceptible T. durum accession D447 (DW1) with its resistant BC3F9 and BC3F10 (B9 and B10) near isogenic lines, which carried Yr15 introgressed from G25. The nearest markers segregated 0.1 cM proximally and 1.1 cM distally to Yr15. These markers were also mapped and validated at the same position in another 500 independent F2 plants derived from crosses of B9 and B10 with the susceptible cultivar Langdon (LDN). SC2700 and SC790, defining Yr15 on an interval of 1.2 cM, were found to be reliable and robust co-dominant markers in a wide range of wheat lines and cultivars with and without Yr15. These markers are useful tags in marker-assisted wheat breeding programs that aim to incorporate Yr15 into elite wheat lines and cultivars for durable and broad-spectrum resistance to stripe rust.

Allele-specific amplification for the detection of ascochyta blight resistance in chickpea

A new co-dominant molecular marker, CaETR, was developed based on allelic sequence length polymorphism in an ethylene receptor-like gene located in the genomic region of a QTL (QTLAR1) conferring ascochyta blight resistance in chickpea. This marker not only discriminated resistance and susceptible phenotypes of chickpea to ascochyta blight, but also easily detected heterozygous genotypes. Using the CaETR marker in combination with a previously developed co-dominant SCAR marker (SCY17590) linked to another QTL (QTLAR2) it was possible to detect resistance alleles in 90 % of resistant accessions in a collection of landraces, advances breeding lines and cultivars, and also detected susceptible alleles in all cases. The results of this study offer a scope for improving the efficiency of conventional chickpea breeding by carrying out negative selection for QTLAR1 and QTLAR2 in early generations without relaying directly on the phenotype. This PCR-based approach using both co-dominant markers is proposed as an efficient tool for selecting blight-resistant genotypes in breeding programs.

Fine mapping of the epistatic suppressor gene (esp) of a recessive genic male sterility in rapeseed (Brassica napus L.)

9012AB, a recessive genic male sterility (RGMS) line derived from spontaneous mutation in Brassica napus, has been playing an important role in rapeseed hybrid production in China. The male sterility of 9012AB is controlled by two recessive genes (ms3 and ms4) interacting with one recessive epistatic suppressor gene (esp). The objective of this study was to develop PCR-based markers tightly linked to the esp gene and construct a high-resolution map surrounding the esp gene. From the survey of 512 AFLP primer combinations, 3 tightly linked AFLP markers were obtained and successfully converted to codominant or dominant SCAR markers. Furthermore, a codominant SSR marker (Ra2G08) associated with the esp gene was identified through genetic map integration. For fine mapping of the esp gene, these PCR-based markers were analyzed in a large BC1 population of 2545 plants. The esp gene was then genetically restricted to a region of 1.03 cM, 0.35 cM from SSR marker Ra2G08 and 0.68 cM from SCAR marker WSC6. The SCAR marker WSC5 co-segregated with the target gene. These results lay a solid foundation for map-based cloning of esp and will facilitate the selection of RGMS lines and their temporary maintainers.

Development of a locus-specific, co-dominant SCAR marker for assisted-selection of the Sw-5 (Tospovirus resistance) gene cluster in a wide range of tomato accessions

Development of a locus-specific, co-dominant SCAR marker for assisted-selection of the Sw-5 (Tospovirus resistance) gene cluster in a wide range of tomato accessions

Development of a locus-specific, co-dominant SCAR marker for assisted-selection of the Sw-5 (Tospovirus resistance) gene cluster in a wide range of tomato accessions

The best levels of broad-spectrum Tospovirus resistance reported in tomatoes thus far are conferred by the Sw-5 locus. This locus contains at least five paralogues (denoted Sw-5a through Sw-5e), of which Sw-5b represents the actual resistance gene. Here we evaluated a panel of seven PCR primer pairs matching different sequences within a genomic region spanning the Sw-5a and Sw-5b gene cluster. Primer efficiency evaluation was done employing tomato isolines with and without the Sw-5 locus. One primer pair produced a single and co-dominant polymorphism between susceptible and resistant isolines. Sequence analysis of these amplicons indicated that they were specific for the Sw-5 locus and their differences were due to insertions/deletions. The polymorphic SCAR amplicon encompass a conserved sequence of the promoter region of the functional Sw-5b gene, being located in the position −31 from its open reading frame. This primer pair was also evaluated in field assays and with a collection of accessions known to be either susceptible or resistant to tospoviruses. An almost complete correlation was found between resistance under greenhouse/field conditions and the presence of the marker. Therefore, this primer pair is a very useful tool in marker-assisted selection systems in a large range of tomato accessions.

Development and fine mapping of three co-dominant SCAR markers linked to the M/m gene in the cucumber plant (Cucumis sativus L.)

Owing to its diverse sex types, the cucumber plant has been studied widely as a model for sex determination. In addition to environmental factors and plant hormones, three major genes-F/f, M/m, and A/a-regulate the sex types in the cucumber plant. By combining the bulked segregant analysis (BSA) and the sequence-related amplified polymorphism (SRAP) technology, we identified eight markers linking to the M/m locus. Among them, the two closely linked SRAP markers flanking the M/m locus were the co-dominant marker ME1EM26 and the dominant marker ME1EM23. Further, the co-dominant marker ME8SA7 co-segregated with the M/m locus. With the chromosome walking method using the cucumber genomic bacterial artificial chromosome (BAC) library, we successfully developed a co-dominant SCAR marker S_ME1EM23 from the ME1EM23 sequence. Along with the other two co-dominant SCAR markers S_ME1EM26 and S_ME8SA7 (developed from ME1EM26 and ME8SA7, respectively) in a larger segregating population (900 individuals), the M/m locus was mapped between S_ME1EM26 (5.4 cM) and S_ME1EM23 (0.7 cM), and S_ME8SA7 co-segregated with it.

Identification of two AFLP markers linked to bacterial wilt resistance in tomato and conversion to SCAR markers

Tomato bacterial wilt (BW) incited by Ralstonia solanacearum is a constraint on tomato production in tropical, subtropical and humid regions of the world. In this paper, we present the results of a research aimed at the identification of PCR-based markers amplified fragment length polymorphism (AFLP) linked to the genes that confer resistance to tomato BW. To this purpose, bulked segregant analysis was applied to an F(2) population segregating for the BW resistant gene and derived from the pair-cross between a BW resistant cultivar T51A and the susceptible cultivar T9230. Genetic analysis indicated that tomato BW was conferred by two incomplete dominant genes. A CTAB method for total DNA extraction, developed by Murray and Thompson with some modifications was used to isolation the infected tomato leaves. Thirteen differential fragments were detected using 256 primer combinations, and two AFLP markers were linked to the BW resistance. Subsequently, the AFLP markers were converted to co-dominant SCAR markers, named TSCAR(AAT/CGA) and TSCAR(AAG/CAT). Linkage analysis showed that the two markers are on the contralateral side of TRSR-1. Genetic distance between TSCAR(AAT/CGA) and TRS-1 was estimated to 4.6 cM, while 8.4 cM between TSCAR(AAG/CAT) and TRS-1.

Development and mapping of a codominant SCAR marker linked to the andromonoecious gene of melon

Monoecy is an important goal for melon breeding because of the agronomic advantages it provides to parental lines in that they do not require hand emasculation to develop monoecious F1 hybrids, the latter producing fruits of higher quality. Monoecious phenotype is conferred by the dominant allele of the andromonoecious (a) gene, whereas recessive homozygous plants are andromonoecious. A bulked segregant analysis (BSA) approach performed in a set of 38 double-haploid lines has allowed us to identify an AFLP marker linked to the a gene at 3.3 cM. Following cloning and sequencing of the AFLP fragment, specific PCR primers were designed and used in the amplification of a codominant SCAR marker. Using a backcrossed mapping population of 530 plants, the SCAR marker could be mapped near the a locus (5.5 cM). Size difference between the two allelic SCAR fragments is 42 bp and might be due to a deletion/insertion. The SCAR marker is closest to the a gene identified to date, and can be useful in breeding programs, using marker-assisted selection procedures to screen for sexual types in melon.

Conversion of AFLP markers to sequence-specific markers for closely related lines in rice by use of the rice genome sequence

DNA polymorphism between two major japonica rice cultivars, Nipponbare and Koshihikari, was identified by AFLP. Eighty-four polymorphic AFLP markers were obtained by analysis with 360 combinations of primer pairs. Nucleotide sequences of 73 markers, 29 from Nipponbare and 44 from Koshihikari, were determined, and 46 AFLP markers could be assigned to rice chromosomes based on sequence homology to the rice genome sequence. Specific primers were designed for amplification of the regions covering the AFLP markers and the flanking sequences. Out of the 46 primer pairs, 44 amplified single DNA fragments, six of which showed different sizes between Nipponbare and Koshihikari, yielding codominant SCAR markers. Eight primer pairs amplified only Nipponbare sequences, providing dominant SCAR markers. DNA fragments amplified by 13 primer pairs showed polymorphism by CAPS, and polymorphism of those amplified by 13 other primer pairs were detected by PCR-RF-SSCP (PRS). Nucleotide sequences of the other four DNA fragments were determined in Koshihikari, but no difference was found between Koshihikari and Nipponbare. In total, 40 sequence-specific markers for the combination of Nipponbare and Koshihikari were produced. All the SNPs identified by AFLP were detectable by CAPS and PRS. The same method was applicable to a combination of Kokoromachi and Tohoku 168, and 23 polymorphic markers were identified using these two rice cultivars. The procedure of conversion of AFLP-markers to the sequence-specific markers used in this study enables efficient sequence-specific marker production for closely related cultivars.

Fine mapping of the parthenocarpic fruit ( pat) mutation in tomato.

The parthenocarpic fruit ( pat) gene of tomato is a recessive mutation conferring parthenocarpy, which is the capability of a plant to set seedless fruits in the absence of pollination and fertilization. Parthenocarpic mutants offer a useful method to regulate fruit production and a suitable experimental system to study ovary and fruit development. In order to map the Pat locus, two populations segregating from the interspecific cross Lycopersicon esculentum x Lycopersicon pennellii were grown, and progeny plants were classified as parthenocarpic or wild-type by taking into account some characteristic aberrations affecting mutant anthers and ovules. Through bulk segregant analysis, we searched for both random and mapped AFLPs linked to the target gene. In this way, the Pat locus was assigned to the long arm of chromosome 3, as also confirmed by the analysis of a set of L. pennellii substitution and introgression lines. Afterwards, the Pat position was refined by using simple sequence repeats (SSRs) and conserved ortholog set (COS) markers mapping in the target region. The tightest COSs were converted into CAPS or SCAR markers. At present, two co-dominant SCAR markers encompassing a genetic window of 1.2 cM flank the Pat locus. Considering that these markers are orthologous to Arabidopsis genes, a positional cloning exploiting the tomato- Arabidopsis microsynteny seems to be a short-term objective.

Molecular characterization of RAPD and SCAR marker linked to the frog-eye leaf spot resistance gene in soybean

Two fragments SCS3620 and SCS3580 of the co-dominant marker OPS03620 & 580 that were linked to the resistance gene of soybean frog-eye leaf spot have been completely sequenced. A significant insertion of 30 bp is the main reason of the polymorphism between the two fragments. The results of Southern hybridization indicate that SCS3620 derives from a single-or low-copy sequence and can be used as an RFLP probe. A co-dominant SCAR marker SCS3620 & 580 has been developed based on the sequences. The segregation of SCS3620 & 580 is similar to that of RAPD marker OPS03620 & 580-Significant polymorphism has been shown between resistant and susceptible genotypes when 62 soybean genotypes were surveyed for the SCAR marker. Therefore, the marker can be used in the resistance breeding of soybean frog-eye leaf spot by marker-assisted selection.

Identification of a codominant scar marker linked to the seedlessness character in grapevine

The variety Vitis vinifera cv Sultanine presents a type of seedlessness in which fertilization occurs but seeds subsequently fail to develop. It has been suggested that this trait might be controlled by three complementary recessive genes regulated by a dominant gene named I. Bulk segregant analysis was used to search for random amplified polymorphic DNA (RAPD) markers linked to the I gene in progeny obtained by crossing two partially seedless genotypes. One hundred and forty decamer primers were screened using bulks obtained by pooling the DNA of extreme individuals from the phenotypic distribution. We identified two RAPD markers which appeared tightly linked to I (at 0.7 and 3.5 cM respectively). The closest marker was used to develop a codominant SCAR (sequence characterized amplified region), named SCC8. This latter marker appeared of great value either to exclude from the progeny potentially seeded individuals or to select for seedless individuals. Indeed, all the seeded individuals of the progeny were found to be homozygous scc8 -/scc8 -, and all the individuals homozygous SCC8 +/SCC8 + were seedless. Moreover, this marker was successfully applied to other natural seedless varieties where codominance persisted. SCC8 was also used to dissect more precisely the genetics of seedlessness. ANOVA analysis indicated that this SCAR marker accounted for at least 64.9% of the phenotypic variation of the seed’s fresh weight and for at least 78.7% of the phenotypic variation of the seed’s dry matter. These results confirmed the presence of a major gene, and also the existence of other complementary recessive genes, controlling the expression of seedlessness.

The role of RAPD markers in breeding for disease resistance in common bean

Diseases are regarded as the leading constraint to increased common bean (Phaseolus vulgaris L.) production worldwide. The range in variability and complexity among bean pathogens can be controlled with different single gene and quantitative resistance sources. Combining these resistance sources into commercial cultivars is a major challenge for bean breeders. To assist breeders, a major effort to identify RAPD markers tightly linked to different genes was undertaken. To date, 23 RAPD and five SCAR markers linked to 15 different resistance genes have been identified, in addition to QTL conditioning resistance to seven major pathogens of common bean. We review the feasibility of using marker-assisted selection (MAS) to incorporate disease resistance into common bean. Indirect selection of single resistance genes in the absence of the pathogen and the opportunity afforded breeders to pyramid these genes to improve their longevity and retain valuable hypostatic genes is discussed. The role of markers linked to the QTL controlling complex resistance and the potential to combine resistance sources using marker based selection is reviewed. Improving levels of selection efficiency using flanking markers, repulsion-phase linkages, co-dominant marker pairs, recombination-facilitated MAS and SCAR markers is demonstrated. Marker-assisted selection for disease resistance in common bean provides opportunities to breeders that were not feasible with traditional breeding methods.