Molecular markers and their applications in wheat breeding

In recent years, considerable emphasis has been placed on the development of molecular markers to be used for a variety of objectives. This review attempts to give an account of different molecular markers—restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), sequence-tagged sites (STS), DNA amplification fingerprinting (DAF), amplified fragment length polymorphisms (AFLPs) and microsatellites (STMS)—currently available for genome mapping and for tagging different traits in wheat. Other markers, including microsatellite-primed polymerase chain reaction (MP-PCR), expressed sequence tags (ESTs) and single nucleotide polymorphisms (SNPs) are also discussed. Recent information on synteny in cereal genomes, marker-assisted selection, marker validation and their relevance to cereal breeding in general and wheat breeding in particular are also examined.     

Utility of AFLP markers for the assessment of genetic diversity within Brassica nigra germplasm

Genetic diversity of 18 Brassica nigra accessions was estimated using amplified fragment length polymorphism (AFLP) marker technology. Two B. rapa and two B. juncea accessions were selected as outliers in the study. Eight AFLP primer combinations generated a total of 426 bands, of which 79% were polymorphic. The UPGMA method was employed to construct a dendrogram based on the Jaccard's similarity coefficient. The accessions of B. rapa separated from those of B. nigra at a genetic similarity coefficient of 0.27 while those of B. juncea did so at 0.5. The genetic similarity coefficients within the B. nigra accessions ranged from 0.58 to 0.86. Based on these coefficients it was concluded that the B. nigra accessions show high levels of genetic variation. These results have significant implications in the crop improvement programmes for the agronomically important crop B. juncea, an amphidiploid of B. nigra and B. rapa. Two incorrectly labelled B. nigra accessions were also identified. These accessions were found to cluster with those of B. juncea accessions. This result demonstrates the great value of AFLP markers in the management of genebanks.     

Genetic diversity of oilseed Brassica napus inbred lines based on sequence-related amplified polymorphism and its relation to hybrid performance

Significant heterosis for seed yield in oilseed rape has created interest in the development of hybrid cultivars. The DNA‐based marker protocol, sequence‐related amplified polymorphism (SRAP) was used to determine genetic diversity among oilseed rape maintainer and restorer lines. This measure was used in an attempt to establish an association between genetic distance and heterosis in hybrids for various agronomic traits. A total of 118 polymorphic loci were generated by 18 SRAP primer combinations. Based on the polymorphism generated by the markers, calculated similarity index values ranged from 0.46 to 0.97. Cluster analysis grouped 10 maintainer and 12 restorer lines into three groups, with the exception of two maintainer lines, PM5 and PM9, which fell outside these groups. The grouping of the lines was largely in agreement with the available pedigree data on their origin and agronomic performance. Analysis of variance among inbred lines and their resulting F₁ hybrids over two locations revealed significant differences for plant height, days to maturity and seed yield, but not for oil content. Substantial mid‐parent heterosis was observed only for seed yield, and ranged from 26% to 169%. All hybrids surpassed their respective inbred lines for this trait, except for a single cross combination of related lines. In general, crosses of lines located in different clusters yielded more than those from the same clusters. Regression analysis revealed a statistically significant relationship between the genetic distance of the parents and seed yield in their hybrid, and their derived mid‐parent and high‐parent heterosis. The correlation coefficient between genetic distance and yield (0.64) indicated a moderately strong relationship, so it is possible that some of the SRAP markers might be linked to quantitative trait loci for seed yield.     

Genetic diversity among populations and breeding lines from recurrent selection in Brassica napus as revealed by RAPD markers

Recurrent selection facilitated by dominant male sterility has been conducted to broaden the genetic basis for cultivar development in Brassica napus. This study aimed to evaluate the genetic variation in four base populations (C0‐C3) and breeding lines from two of the populations produced during recurrent selection by random amplified polymorphic DNA (Rapd) markers. Genetic variation in four populations declined gradually with the advance of selection cycles as measured by expected genetic heterozygosity (from 0.2058 in C0 to 0.1536 in C3) but the decline was not statistically significant. When compared with the average genetic distances for 21 germplasm collections with wide geographical and genetic origins (0.4712) and seven breeding lines from pedigree selection (0.2059), seven breeding lines selected from the C1 population and 11 from the C3 population had a larger average genetic distance (0.5339 and 0.5486, respectively). Clustering analysis indicated that the lines from recurrent selection had a much lower genetic similarity than lines from pedigree selection. Our results suggest that base populations derived from recurrent selection could provide a wider genetic variation for selection of breeding lines with more broad genetic bases.     

Present and future of quantitative trait locus analysis in plant breeding

The joint analysis of genotype marker segregation and phenotypic values of individuals or lines enables the detection and location of loci affecting quantitative traits (QTL). The availability of DNA markers and powerful biometric methods has led to considerable progress in QTL mapping in plants. The most obvious applications of QTL analysis seem to be marker‐assisted selection (MAS) in breeding and pre‐breeding and QTL cloning. However, other areas are envisaged where QTL analysis can contribute decisively. These are: the understanding of complex traits such as plant‐pathogen interaction; plant genomics, connecting proteins and regulatory elements of known functions to QTL by candidate gene analysis; and germplasm enhancement through a characterization that allows its efficient utilization. The success in all these applications depends primarily on the reliability and accuracy of the QTL analysis itself. Therefore, the discussion of its limitations will constitute an important part of this review.     

Molecular mapping of a dominant genic male sterility gene Ms in rapeseed (Brassica napus)

Rs1046AB is a genic male sterile two‐type line in rapeseed that has great potential for hybrid seed production. The sterility of this line is conditioned by the interaction of two genes, i.e. the dominant genic male sterility gene (Ms) and the suppressor gene (Rf). The present study was undertaken to identify DNA markers for the Ms locus in a BC₁ population developed from a cross between a male‐sterile plant in Rs1046AB and the fertile canola‐type cultivar ‘Samourai’. Bulked segregant analysis was performed using the amplified fragment length polymorphism (AFLP) methodology. From the survey of 480 AFLP primer combinations, five AFLP markers (P10M13₃₅₀, P13M8₄₀₀, P6M6₄₁₀, E7M1₂₃₀ and E3M15₁₀₀) tightly linked to the target gene were identified. Two of them, E3M15₁₀₀ and P6M6₄₁₀, located the closest, at either side of Ms at a distance of 3.7 and 5.9 cM, respectively. The Ms locus was subsequently mapped on linkage group LG10 in the map developed in this laboratory, adding two additional markers weakly linked to it. This suite of markers will be valuable in designing a marker‐assisted genic male sterility three‐line breeding programme.     

Mapping of QTL controlling tocopherol content in winter oilseed rape

Tocopherols are natural antioxidants in vegetable oils and are important dietary nutrients. Enhanced tocopherol content has become an important objective in oilseed rape breeding. A segregating DH population was tested for 2 years at two locations in replicated field trials. Genotypic differences occurred for α‐, γ‐ and total tocopherol content as well as α/γ‐tocopherol ratio, but highly significant genotype x environment interactions resulted in low heritabilities. Using a mixed‐model composite interval mapping approach between one and five QTL with additive and/or additive x environment interaction effects could be mapped for α‐, γ‐ and total tocopherol content and α/β‐tocopherol ratio. In addition, one to six locus pairs with epistatic interaction effects were identified, indicating a strong contribution of epistasis to trait variation. In total, the additive and epistatic effects explained between 28% (α‐tocopherol content) and 73% (total tocopherol content) of the genotypic variance in the population, with individual QTL and locus pairs contributing between 7.5 and 29.2% of variance. Considering the low heritabilities of the tocopherol traits, the results of this study indicate that marker‐assisted selection may be an efficient strategy in a breeding program for enhanced tocopherol content in rapeseed.     

Molecular markers: actual and potential contributions to wheat genome characterization and breeding

The demands for increasing global crop production have prompted the development of new approaches relying on molecular marker technologies to investigate and improve the plant genome. The merits of molecular markers make them valuable tools in a range of research areas. This review describes novel approaches based on modern molecular marker technologies for characterization and utilization of genetic variation for wheat improvement. Large-scale genome characterization by DNA-fingerprinting has revealed no declining trends in the molecular genetic diversity in wheat as a consequence of modern intensive breeding thus opposing the genetic ‘erosion’ hypothesis. A great number of important major genes and quantitative trait loci have been mapped with molecular markers. Marker-assisted selection based on a tight linkage between a marker allele and a gene(s) governing a qualitative or quantitative trait is gaining considerable importance as it facilitates and accelerates cultivar improvement through precise transfer of chromosome regions carrying the gene of interest. The implementations of molecular markers in wheat genotyping, mapping and breeding complemented by specific approaches associated with the complex polyploid nature of common wheat are analyzed and presented.     

Identification of AFLP markers linked to the epistatic suppressor gene of a recessive genic male sterility in rapeseed and conversion to SCAR markers

A recessive epistatic genic male sterility (REGMS) two‐type line, 9012AB, has been used for rapeseed hybrid seed production in China. The male sterility of 9012AB is controlled by two recessive duplicate sterile genes (ms1 and ms2) interacting with one recessive epistatic suppressor gene (esp). Homozygosity at the esp locus (espesp) suppresses the expression of the recessive male sterility trait in homozygous ms1ms1ms2 ms2 plants. In this study, we used a combination of bulked segregant analyses and amplified fragment length polymorphism (AFLP) to identify markers linked to the suppressor gene in a BC₁ population. From the survey of 1024 AFLP primer combinations, eight markers tightly linked to the target gene were identified. The two closest markers flanking both sides of Esp, P9M5₃₇₀ and S16M14₇₈₀, had a genetic distance of 1.4 cM and 2.1 cM, respectively. The AFLP fragment from P4M8₁₉₀, which co‐segregated with the target gene was converted into a sequence characterized amplified region marker. The availability of linked molecular markers will facilitate the utilization of REGMS in hybrid breeding in Brassica napus.     

分子标记在根瘤菌的运用

摘要: 分子标记的发展为根瘤菌的研究带来了重大突破,应用于根瘤菌遗传图谱构建,遗传多态性及遗传区域分析,品种鉴别等各个方面。本文主要介绍RFLP RAPD SSR ISSR等的分子标记原理、方法特点以及在根瘤菌分类上的应用。     

Linkage of random amplified polymorphic DNA markers to downy mildew resistance in cucumber (Cucumis sativus L.)

Marker assisted selection (MAS) may improve the efficiency of breeding downy mildew resistant cucumber cultivars. A study was conducted to identify random amplified polymorphic DNA (RAPD) markers linked to the downy mildew resistance gene (dm) which would be suitable for MAS. A total of 145 F₃ families from two populations (55 from the WI 1983G × Straight 8 population and 90 from the Zudm1 × Straight 8 population) were evaluated over five locations in North America and Europe. Resistant and susceptible F₃ families were identified and mean family resistance ratings were used to type individual F₂ plants. No evidence for race differences in the pathogen (Psuedoperonospora cubensis (Berk. & Curt.) Rostow) between North America and Europe was found. Phenotypic correlations between locations ranged from 0.3 to 0.7. Of the 135 polymorphic RAPD markers identified from 960 primers, five were linked to dm - G14₈₀₀, X15₁₁₀₀, AS5₈₀₀, BC519₁₁₀₀, and BC526₁₀₀₀. In the WI 1983G × Straight 8 population, G14₈₀₀ was linked to dm at 16.5 cm, AS5₈₀₀ at 32.8 cm, BC519₁₁₀₀ at 9.9 cm, and BC526₁₀₀₀ at 19.2 cm. In the Zudm1 ×Straight 8 population, G14₈₀₀ was linked at 20.9 cm, X15₁₁₀₀at 14.8 cm, AS5₈₀₀ at 24.8 cm, and BC526_{_1000} at 32.9 cm. MarkersG14₈₀₀ and BC519₁₁₀₀ were linked in repulsion to the dm allele, and X15₁₁₀₀, AS5₈₀₀, and BC526₁₀₀₀ were linked in coupling phase. These genetic markers may be exploited to develop an efficient MAS strategy for breeding resistant cucumber cultivars. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of AFLP and RAPD markers linked to anthracnose resistance in grapes and their conversion to SCAR markers

Resistance to anthracnose or black spot ( Elsinoe ampelina ), a serious fungal pathogen in viticulture and table grape production, was investigated on 25 grape cultivars. Bioassays performed with culture filtrates produced by the pathogen revealed 14 resistant genotypes. In most plants resistance originated from Vitis labrucsa but also genotypes with V. rupestris and V. riparia  ×  V. rupestris background showed resistance. Genetic analysis was conducted in F₁, S₁ and BC₁ plants developed from various cultivars. In total, 326 F₁ plants were evaluated, 172 genotypes proofed to be resistant, whereas 154 were susceptible to anthracnose. A Mendelian segregation ratio of 1 : 1 (χ² = 0.30–0.65) indicating that anthracnose resistance is controlled by a single dominant gene. To facilitate the use of marker-assisted selection in grape-breeding PCR-based markers were developed by random amplified polymorphic DNA and amplified fragment length polymorphism in bulk segregant analysis. Finally, OPB 15₁₂₄₇ was developed as a sequence characterized amplified region marker being diagnostic for the locus of resistance to anthracnose in all resistant genotypes tested. Within the 25 grape cultivars OPB 15₁₂₄₇ is diagnostic in the genetic background of both V. labrucsa and V. rupestris and V. riparia  ×  V. rupestris .     

Molecular assessment of genetic diversity in winter barley and its use in breeding

Summary During the last decades extensive progress has been achieved in winter barley breeding with respect to both, yield and resistance to fungal and viral diseases. This progress is mainly due to the efficient use of the genetic diversity present within high yielding adapted cultivars and – with respect to resistance – to the extensive evaluation of genetic resources followed by genetic analyses and introgression of respective genes by sexual recombination. Detailed knowledge on genetic diversity present on the molecular level regarding specific traits as well as on the whole genome level may enhance barley breeding today by facilitating efficient selection of parental lines and marker assisted selection procedures. In the present paper the state of the art with respect to virus diseases, i.e. Barley mild mosaic virus, Barley yellow mosaic virus, and Barley yellow dwarf virus is briefly reviewed and first results on a project aiming on a genome wide estimation of genetic diversity which in combination with data on yield and additional agronomic traits may facilitate the detection of marker trait associations and a more efficient selection of parental genotypes are presented. By field tests of 49 two-rowed and 64 six-rowed winter barley cultivars the genetic gain in yield for the period 1970–2003 was estimated at 54.6 kg ha⁻¹ year⁻¹ (r² = 0.567) for the six-rowed cultivars and at 37.5 kg ha⁻¹ year⁻¹ (r² = 0.621) for the two-rowed cultivars. Analysis of 30 SSRs revealed a non-homogenous allele distribution between two and six-rowed cultivars and changes of allele frequencies in relation to the time of release. By PCoA a separation between two and six-rowed cultivars was observed but no clear cut differentiation in relation to the time of release. In the two-rowed cultivars an increase in genetic diversity (DI) from older to newly released cultivars was detected.     

Mapping of a gene for leaf scald resistance in barley line 'B87/14' and validation of microsatellite and RFLP markers for marker-assisted selection

Seedlings of the barley line ‘B87/14’ were resistant to 22 out of 23 Australian isolates of Rhynchosporium secalis, the causal agent of leaf scald.‘B87/14’‐based populations were developed to determine the location of the resistance locus. Scald resistance segregated as a single dominant trait in BC₁F₂ and BC₁F₃ populations. Bulked segregant analysis identified amplified fragment length polymorphisms (AFLPs) with close linkage to the resistance locus. Fully mapped populations not segregating for scald resistance located these AFLP markers on chromosome 3H, possibly within the complex Rrs1 scald locus. Microsatellite and restriction fragment length polymorphism markers adjacent to the AFLP markers were identified and validated for their linkage to scald resistance in a second segregating population, with the closest marker 2.2 cM from the resistance locus. These markers can be used for selection of the Rrs.B87 scald‐resistance locus, and other genes at the chromosome 3H Rrs1 locus.     

Genetic relationships between Sicilian wild populations of Brassica analysed with RAPD markers

Brassica comprises very variable species, both morphologically and genetically. Among these species, the Sicilian populations of Brassica sect. Brassica, species related to kale crops form a complex group. The genetic relationships among 15 populations occurring in Sicily and one from Calabria, representing the existing diversity, have been investigated using random amplified polymorphic DNA (RAPD) markers. This assay, carried out with 22 arbitrary primers, generated 236 polymorphic fragments, 21 of which were specific for single populations (mainly Brassica insularis, Brassica incana and Brassica macro‐carpa). Jaccard's genetic distances were computed and the phylogenic tree was established using the UPGMA algorithm. The dendrogram obtained showed four branches grouping: (1) B. incana populations; (2) B. insularis and B. macrocarpa, occurring in small islands around Sicily; (3) B. rupestris populations; and (4) B. villosa populations. Within B. rupestris, only B. rupestris subsp. brevisiliqua was clustered in the B. villosa group. The classification obtained is discussed with regard to the morphological, ecological and geographical data.     

Molecular genetics and breeding of vegetable brassicas

Summary Genetic traits relevant to breeding vegetable brassicas are surveyed to determine the effects that recent advances in molecular genetics and molecular marker technology might have on breeding priorties and practices. The problems peculiar to breeding horticultural crops are addressed. The application of DNA restriction fragment length polymorphisms (RFLPs) as an aid to breeding brassicas is discussed and genes amenable to molecular analysis are considered in the light of both experimental and economic factors.     

Molecular markers for yield components in Brassica juncea – do these assist in breeding for high seed yield?

A population of 112 F₁-derived doubled haploid lines was produced from a reciprocal cross of Brassica juncea. The parents differed for seed quality, seed color and many agronomic traits. A detailed RFLP linkage map of this population, comprising 316 loci, had been constructed, and was used to map quantitative trait loci (QTL) for seed yield and yield components, viz. siliqua length, number of seeds per siliqua, number of siliques per main raceme and 1000-seed weight. Stable and significant QTLs were identified for all these yield components except seed yield. For yield components, a selection index based on combined phenotypic and molecular data (QTL effects) could double up the efficiency of selection compared to the expected genetic advance by phenotypic selection. Selection indices for high seed yield, based on the phenotypic data of yield and yield components, could only improve the efficiency of selection by 4% of the genetic advance that can be expected from direct phenotypic selection for yield alone. Inclusion of molecular data together with the phenotypic data of yield components in the selection indices did not improve the efficiency of selection for higher seed yield. This is probably due to often negative relationships among the yield components. Most of the QTLs for yield components were compensating each other, probably due to linkage, pleiotropy or developmentally induced relationships among them. The breeding strategy for B. juncea and challenges to marker assisted selection are discussed.     

Molecular advancements in male sterility systems of Capsicum: A review

In recent years, plant molecular research on genetic mapping, gene tagging and cloning, and marker-assisted selection (MAS) have gained importance in crop improvement programmes. In Capsicum, several inter- and intra-specific genetic maps with wide distribution of markers covering the whole genome have been developed. Recently, whole genome of the hot pepper C. annuum, its wild progenitor C. annuum var. glabriusculum and C. baccatum has been sequenced. The Capsicum genome size has been estimated to be approx. 4× (3.48 Gb) the genome size of cultivated tomato (Solanum lycopersicum L.) (900 Mb). Breeders’ access to the pepper genomic information would facilitate the choice of markers from different linkage groups, thus paving the way for gene cloning and its introgression into the elite breeding lines through MAS. Till date, approx. 20 independently inherited nuclear male sterility (NMS) genes have been reported. Linked markers have been identified for ms1, ms3, ms8, ms10, ms_k, msc-1 and an undesignated gene. However, markers tightly linked to ms8 and ms10 are still lacking. Except ms1, ms3, ms8 and ms10, the map position of other NMS genes is not known. In cytoplasmic male sterility (CMS), markers for the mitochondrial gene atp6 have been developed and the gene cloned. Number of markers some very tightly linked to the restorer-of-fertility (Rf) gene have been identified. However, the actual map position of the Rf locus is still not determined. Another CMS-associated nuclear gene “pr” responsible for restoring partial fertility has been identified and tagged. In this review, we have compiled up-to-date information about the marker technology relating to the NMS and the CMS-associated genes in Capsicum. This information can be useful when screening Capsicum germplasm, developing NMS lines through MAS, improving efficiency of the NMS system, transferring rf gene for maintainer line breeding and Rf genes for restorer line breeding in CMS and assessing genetic purity of the hybrid seed.     

Genetics,genomics and breeding of groundnut (Arachis hypogaea L.)

Groundnut is an important food and oil crop in the semiarid tropics, contributing to household food consumption and cash income. In Asia and Africa, yields are low attributed to various production constraints. This review paper highlights advances in genetics, genomics and breeding to improve the productivity of groundnut. Genetic studies concerning inheritance, genetic variability and heritability, combining ability and trait correlations have provided a better understanding of the crop's genetics to develop appropriate breeding strategies for target traits. Several improved lines and sources of variability have been identified or developed for various economically important traits through conventional breeding. Significant advances have also been made in groundnut genomics including genome sequencing, marker development and genetic and trait mapping. These advances have led to a better understanding of the groundnut genome, discovery of genes/variants for traits of interest and integration of marker‐assisted breeding for selected traits. The integration of genomic tools into the breeding process accompanied with increased precision of yield trialing and phenotyping will increase the efficiency and enhance the genetic gain for release of improved groundnut varieties.