The successful application of a marker-assisted wheat breeding strategy

A number of useful marker-trait associations have been reported for wheat. However the number of publications detailing the integrated and pragmatic use of molecular markers in wheat breeding is limited. A previous report by some of these authors showed how marker-assisted selection could increase the genetic gain and economic efficiency of a specific breeding strategy. Here, we present a practical validation of that study. The target of this breeding strategy was to produce wheat lines derived from an elite Australian cultivar ‘Stylet’, with superior dough properties and durable rust resistance donated from ‘Annuello’. Molecular markers were used to screen a BC₁F₁ population produced from a cross between the recurrent parent ‘Stylet’ and the donor parent ‘Annuello’ for the presence of rust resistance genes Lr34/Yr18 and Lr46/Yr29. Following this, marker-assisted selection was applied to haploid plants, prior to chromosome doubling with cochicine, for the rust resistance genes Lr24/Sr24, Lr34/Yr18, height reducing genes, and for the grain protein genes Glu-D1 and Glu-A3. In general, results from this study agreed with those of the simulation study. Genetic improvement for rust resistance was greatest when marker selection was applied on BC₁F₁ individuals. Introgression of both the Lr34/Yr18 and Lr46/Yr29 loci into the susceptible recurrent parent background resulted in substantial improvement in leaf rust and stripe rust resistance levels. Selection for favourable glutenin alleles significantly improved dough resistance and dough extensibility. Marker-assisted selection for improved grain yield, through the selection of recurrent parent genome using anonymous markers, only marginally improved grain yield at one of the five sites used for grain yield assessment. In summary, the integration of marker-assisted selection for specific target genes, particularly at the early stages of a breeding programme, is likely to substantially increase genetic improvement in wheat.     

Marker-assisted selection and evaluation of high oil in vivo haploid inducers in maize

Doubled haploid technology, which is used to rapidly purify genetic resources, is one of the key technologies in modern maize breeding. In a previous study, the major quantitative trait locus qhir1, which influences in vivo haploid induction, was narrowed down to a 243-kb region, which made it feasible to use marker-assisted selection (MAS) for inducer development. Recently, a new method was developed for haploid identification using oil content (OC). The objective of this study was to develop high oil inducer lines using MAS of the qhir1 locus. We constructed an F₂ population, two backcross populations that were backcrossed to the inducer CAU5 (BC₁F₁-CAU5) and the high oil inbred line GY923 (BC₁F₁-GY923), respectively, which was derived from the cross GY923 × CAU5, and subjected continuous selfing to develop high oil inducer lines. In each cycle, three different parameters including kernel OC, marker genotype at qhir1 and haploid induction rate (HIR) were used for pedigree selection. Three candidate high oil inducer lines were developed, with an OC of approximately 8.5 %, an HIR of approximately 8 % and superior agronomic performance, which are suitable values for the application of these lines to haploid identification by OC. Our results confirm the notion that HIR selection combined with MAS for qhir1 is an effective approach to haploid inducer breeding. In addition, we determined that the accuracy of haploid identification by OC is influenced by the female germplasm resource and the high oil inducer and that appropriate critical points for OC can balance the false discovery rate and false negative rate.     

Coupling- and repulsion-phase RAPDs for marker-assisted selection of PI 181996 rust resistance in common bean

The Guatemalan black bean (Phaseolus vulgaris L.) plant introduction (PI) 181996 is resistant to all known US races of the bean rust fungus Uromyces appendiculatus (Pers. ex Pers.) Unger var. appendiculatus [syn. U. phaseoli (Reben) Wint.]. We report on two random amplified polymorphic DNA (RAPD) markers OAC20₄₉₀ tightly linked (no recombinants) in coupling phase and OAE19₈₉₀ linked in repulsion phase (at 6.2±2.8 cM) to PI 181996 rust resistance. These RAPDs, generated by single decamer primers in the polymerase chain reaction, were identified in near-isogenic bulks of non-segregating resistant and susceptible BC₄F₂ (NX-040*4/PI 181996) lines. Linkage of the RAPD markers was confirmed by screening 19 BC₄F₂ and 57 BC₄F₃ individuals segregating for PI 181996 resistance. Utility of the RAPDs OAC20₄₉₀ and OAE19₈₉₀ was investigated in a diverse group of common bean cultivars and lines. All cultivars into which the PI 181996 resistance was introgressed had the RAPD OAC20₄₉₀. A RAPD similar in size to OAC20₄₉₀, observed in some susceptible common bean lines, was confirmed by Southern blotting to be homologous to the RAPD OAC20₄₉₀. Use of the RAPDs OAC20₄₉₀ and OAE19₈₉₀ in marker-assisted selection (MAS) is proposed. The coupling-phase RAPD is most useful for MAS of resistant BC_nF₁individuals during traditional backcross breeding. The repulsion-phase RAPD has greatest utility in MAS of homozygous-resistant individuals in F₂ or later-segregating generations.Mention of a trademark or a proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable.     

Combining bacterial blight resistance and Basmati quality characteristics by phenotypic and molecular marker-assisted selection in rice

Bacterial Blight (BB) caused by Xanthomonas oryzae pv. oryzae is a major disease of rice in tropical Asia. Since all the Basmati varieties are highly susceptible and the disease is prevalent in the entire Basmati growing region of India, BB is a severe constraint in Basmati rice production. The present study was undertaken with the objective of combining the important Basmati quality traits with resistance to BB by a combination of phenotypic and molecular marker-assisted selection (MAS). Screening of 13 near-isogenic lines of rice against four isolates of the pathogen from Basmati growing regions identified the Xa4, xa8, xa13 and Xa21 effective against all the isolates tested. Two or more of these genes in combination imparted enhanced resistance as expressed by reduced average lesion length in comparison to individual genes. The two-gene pyramid line IRBB55 carrying xa13 and Xa21 was found equally effective as three/four gene pyramid lines. The two BB resistance genes present in IRBB55 were combined with the Basmati quality traits of Pusa Basmati-1 (PB-1), the most popular high yielding Basmati rice variety used as recurrent parent. Phenotypic selection for disease resistance, agronomic and Basmati quality characteristics and marker-assisted selection for the two resistance genes were carried out in BC₁F₁, BC₁F₂ and BC₁F₃ generations. Background analysis using 252 polymorphic amplified fragment length polymorphism (AFLP) markers detected 80.4 to 86.7% recurrent parent alleles in BC₁F₃ selections. Recombinants having enhanced resistance to BB, Basmati quality and desirable agronomic traits were identified, which can either be directly developed into commercial varieties or used as immediate donors of BB resistance in Basmati breeding programs.     

Enhanced tolerance to boron toxicity in two-rowed barley by marker-assisted introgression of favourable alleles derived from Sahara 3771

Boron (B) is an essential micronutrient for higher plant, but toxic levels can seriously diminish grain yield in cereal crops by affecting root growth, and thus restricting water extraction from the subsoil. Amelioration of high concentrations in soils is expensive and not always feasible, so breeding for B tolerance is the most viable alternative. This article reports the marker-assisted (MAS) transfer of favourable alleles from an unadapted six-rowed barley (Hordeum vulgare L.) variety, Sahara 3771, into two-rowed lines adapted to southern Australia. During the backcrossing process, the SSR marker, EBmac679, located on chromosome 4H was used to control the target region in foreground selection, but no background selection was applied. Gene introgression was confirmed with 40 BC₆F₁-derived doubled haploid lines segregating for the SSR marker EBmac679. We used a combination of molecular and conventional assays to unequivocally classify the 40 BC₆F₁-derived DH lines as B tolerant or sensitive, and then compared their means for grain yield measured over 2 years and four locations. Results showed modest improvements in grain yield of lines carrying B tolerance genes at some B toxic environments, and negative impact at others. Our results also showed that malting quality profile was not adversely affected through the introgression of the B tolerance allele from Sahara 3771, allowing the newly developed material to be used by breeding programs without risk of a penalty on malt quality.     

A strategy to develop molecular markers applicable to a wide range of crosses for marker assisted selection in plant breeding: a case study on anthracnose disease resistance in lupin (Lupinus angustifolius L.)

A key challenge in marker-assisted selection (MAS) for molecular plant breeding is to develop markers linked to genes of interest which are applicable to multiple breeding populations. In this study representative F₂ plants from a cross Mandalup (resistant to anthracnose disease) × Quilinock (susceptible) of Lupinus angustifolius were used in DNA fingerprinting by Microsatellite-anchored Fragment Length Polymorphism (MFLP). Nine candidate MFLP markers linked to anthracnose resistance were identified, then ‘validated’ on 17 commercial cultivars. The number of “false positives” (showing resistant-allele band but lack of the R gene) for each of the nine candidate MFLP markers on the 17 cultivars ranged from 1 to 9. The candidate marker with least number of false positive was selected, sequenced, and was converted into a co-dominant, sequence-specific, simple PCR based marker suitable for routine implementation. Testing on 180 F₂ plants confirmed that the converted marker was linked to the R gene at 5.1 centiMorgan. The banding pattern of the converted marker was consistent with the disease phenotype on 23 out of the 24 cultivars. This marker, designated “AnManM1”, is now being used for MAS in the Australian lupin breeding program. We conclude that generation of multiple candidate markers, followed by a validation step to select the best marker before conversion to an implementable form is an efficient strategy to ensure wide applicability for MAS.     

Introduction of bacterial blight resistance into Triguna,a high yielding,mid-early duration rice variety

Bacterial blight (BB) is a serious disease of rice in India. We have used molecular marker-assisted selection in a backcross breeding program to introgress three genes (Xa21, xa13, and xa5) for BB resistance into Triguna, a mid-early duration, high yielding rice variety that is susceptible to BB. At each generation in the backcross program, molecular markers were used to select plants possessing these resistance genes and to select plants that have maximum contribution from the Triguna genome. A selected BC3F1 plant was selfed to generate homozygous BC₃F₂ plants with different combinations of BB resistance genes. Plants containing the two-gene combination, Xa21 and xa13, were found to exhibit excellent resistance against BB. Single plant selections for superior agronomic characteristics were performed on the progeny of these plants, from BC₃F₃ generation onwards. The selected plants were subjected to yield trials at the BC₃F₈ generation and were found to have a significant yield advantage over Triguna. The newly developed lines are being entered into national multi-location field trials. This work represents a successful example of the application of molecular marker-assisted selection for BB resistance breeding in rice.     

Population development by phenotypic selection with subsequent marker-assisted selection for line extraction in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Cucumber (Cucumis sativus L.; 2n=2x=14) has a narrow genetic base, and commercial yield of US processing cucumber has plateaued in the last 15 years. Yield may be increased by altering plant architecture to produce unique early flowering (days to flower, DTF), female (gynoecious, GYN), highly branched (multiple lateral branching, MLB), long-fruited (length:diameter ratio, L:D) cultivars with diverse plant statures. The genetic map position of QTL conditioning these quantitatively inherited yield component traits is known, and linked molecular markers may have utility in marker-assisted selection (MAS) programs to increase selection efficiency, and effectiveness. Therefore, a base population (C₀), created by intermating four unique but complementary lines, was subjected to three cycles (C₁–C₃) of phenotypic (PHE) mass selection for DTF, GYN, MLB, and L:D. In tandem, two cycles of marker-assisted backcrossing for these traits began with selected C₂ progeny (C_2S) to produce families (F₁[i.e., C_2S × C_2S], and BC₁ [i.e., F₁ × C_2S]) for line extraction, and for comparative analysis of gain from selection by PHE selection, and MAS. Frequencies of marker loci were used to monitor selection-dependent changes during PHE selection, and MAS. Similar gain from selection was detected as a result of PHE selection, and MAS for MLB (~0.3 branches/cycle), and L:D (~0.1 unit increase/cycle) with concomitant changes in frequency at linked marker loci. Although genetic gain was not realized for GYN during PHE selection, the percentage of female flowers of plants subjected to MAS was increased (5.6–9.8% per cycle) depending upon the BC₁ population examined. Selection-dependent changes in frequency were also detected at marker loci linked to female sex expression during MAS. MAS operated to fix favorable alleles that were not exploited by PHE selection in this population, indicating that MAS could be applied for altering plant architecture in cucumber to improve its yield potential. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby, marked advertisement solely to indicate this fact. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable.     

Mapping of a Novel Race Specific Resistance Gene to Phytophthora Root Rot of Pepper (Capsicum annuum) Using Bulked Segregant Analysis Combined with Specific Length Amplified Fragment Sequencing Strategy

Phytophthora root rot caused by Phytophthora capsici (P. capsici) is a serious limitation to pepper production in Southern China, with high temperature and humidity. Mapping PRR resistance genes can provide linked DNA markers for breeding PRR resistant varieties by molecular marker-assisted selection (MAS). Two BC₁ populations and an F₂ population derived from a cross between P. capsici-resistant accession, Criollo de Morelos 334 (CM334) and P. capsici-susceptible accession, New Mexico Capsicum Accession 10399 (NMCA10399) were used to investigate the genetic characteristics of PRR resistance. PRR resistance to isolate Byl4 (race 3) was controlled by a single dominant gene, PhR10, that was mapped to an interval of 16.39Mb at the end of the long arm of chromosome 10. Integration of bulked segregant analysis (BSA) and Specific Length Amplified Fragment sequencing (SLAF-seq) provided an efficient genetic mapping strategy. Ten polymorphic Simple Sequence Repeat (SSR) markers were found within this region and used to screen the genotypes of 636 BC₁ plants, delimiting PhR10 to a 2.57 Mb interval between markers P52-11-21 (1.5 cM away) and P52-11-41 (1.1 cM). A total of 163 genes were annotated within this region and 31 were predicted to be associated with disease resistance. PhR10 is a novel race specific gene for PRR, and this paper describes linked SSR markers suitable for marker-assisted selection of PRR resistant varieties, also laying a foundation for cloning the resistance gene.     

Molecular tagging and mapping of the erect panicle gene in rice

Erect panicle (EP) is one of the more important traits of the proposed ideotype of high-yielding rice. Several rice cultivars with the EP phenotype, which has been reported to be controlled by a dominant gene, have been successfully developed and released for commercial production in North China. To analyze the inheritance of the EP trait, we generated segregating F₂ and BC₁F₁ populations by crossing an EP-type variety, Liaojing 5, and a curved-panicle-type variety, Fengjin. Our results confirmed that a dominant gene controls the EP trait. Simple-sequence repeat (SSR) and bulked segregant analyses of the F₂ population revealed that the EP gene is located on chromosome 9, between two newly developed SSR markers, RM5833-11 and RM5686-23, at a genetic distance of 1.5 and 0.9 cM, respectively. Markers closer to the EP gene were developed by amplified fragment length polymorphism (AFLP) analysis with 128 AFLP primer combinations. Three AFLP markers were found to be linked to the EP gene, and the nearest marker, E-TA/M-CTC₂₀₀, was mapped to the same location as SSR marker RM5686-23, 1.5 cM from the EP gene. A local map around the EP gene comprising nine SSR and one AFLP marker was constructed. These markers will be useful for marker-assisted selection (MAS) for the EP trait in rice breeding programs.     

Marker-assisted wheat breeding: present status and future possibilities

Wheat production and productivity in the past witnessed a remarkable growth. However, this growth rate could not be sustained during the last decade, causing concern among world wheat community. Marker-assisted selection (MAS), which is being practiced for improvement of a variety of traits in wheat around the world, may at least partly help in providing the desired solution. Marker-trait associations are now known for a number of simple, but difficult-to-score traits, so that MAS has been found useful for improvement of several of these important economic traits. Breeding strategies including marker-assisted backcrossing, forward breeding, MAS involving doubled haploid technology and F₂ enrichment have been successfully utilized for this purpose. However, for improvement of complex polygenic traits, newer technologies based on high throughput genotyping associated with new marker systems (e.g., DArT and SNP), and new selection strategies such as AB-QTL, mapping-as-you-go, marker-assisted recurrent selection and genome-wide selection will have to be tried in future. The progress made in all these aspects of marker-assisted wheat breeding, and the limitations and future prospects of this emerging technology have been reviewed in this article.     

Selection strategies for the development of rye introgression libraries

Computer simulations can be employed to find optimal procedures for developing introgression libraries in rye with marker-assisted backcrossing. Our objectives were to investigate the effects of the employed (1) breeding scheme, (2) selection strategy, and (3) population sizes on the donor genome coverage of the library, the number of introgression lines carrying additional donor chromosome segments outside the target regions, and the number of required marker data points. With respect to these target criteria, a BC₃S₂ breeding scheme and increasing population sizes from early to advanced generations were superior to a BC₂S₃ breeding scheme and constant population sizes. The smallest number of donor segments outside the target regions was reached with a three-stage selection strategy, which consists on selection for the target segment, selection for recombination at flanking markers and selection for recurrent parent alleles across the entire genome. Omitting the selection for flanking markers in generation BC₁ reduced considerably the number of required marker data points. A pre-selection of chromosomes consisting completely of donor genome in BC₁ was advantageous, if the effort in the breeding nursery should kept minimum. Adopting the described designs can help rye breeders to successfully develop introgression libraries.     

Integrating marker-assisted background analysis with foreground selection for pyramiding bacterial blight resistance genes into Basmati rice

Bacterial leaf blight (BB), caused by the bacterium Xanthomonas oryzae pv. Oryzae (Xoo), is the major constraint amongst rice diseases in India. CSR-30 is a very popular high-yielding, salt-tolerant Basmati variety widely grown in Haryana, India, but highly susceptible to BB. In the present study, we have successfully introgressed three BB resistance genes (Xa21, xa13 and xa5) from BB-resistant donor variety IRBB-60 into the BB-susceptible Basmati variety CSR-30 through marker-assisted selection (MAS) exercised with stringent phenotypic selection without compromising the Basmati traits. Background analysis using 131 polymorphic SSR markers revealed that recurrent parent genome (RPG) recovery ranged up to 97.1% among 15 BC₃F₁ three-gene-pyramided genotypes. Based on agronomic evaluation, BB reaction, aroma, percentage recovery of RPG, and grain quality evaluation, four genotypes, viz., IC-R28, IC-R68, IC-R32, and IC-R42, were found promising and advanced to BC₃F₂ generation.     

Introgression of heat shock protein (Hsp70 and sHsp) genes into the Malaysian elite chilli variety Kulai (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) through the application of marker-assisted backcrossing (MAB)

Backcrossing together with simple sequence repeat marker strategy was adopted to improve popular Malaysian chilli Kulai (Capsicum annuum L.) for heat tolerance. The use of molecular markers in backcross breeding and selection contributes significantly to overcoming the main drawbacks such as increase linkage drag and time consumption, in the ancient manual breeding approach (conventional), and speeds up the genome recovery of the recurrent parent. The strategy was adopted to introgress heat shock protein gene(s) from AVPP0702 (C. annuum L.), which are heat-tolerant, into the genetic profile of Kulai, a popular high-yielding chilli but which is heat sensitive. The parents were grown on seed trays, and parental screening was carried out with 252 simple sequence repeat markers. The selected parents were crossed and backcrossed to generate F₁ hybrids and backcross generations. Sixty-eight markers appeared to be polymorphic and were used to assess the backcross generation; BC₁F₁, BC₂F₁ and BC₃F₁. The average recipient allele of the selected four BC₁F₁ plants was 80.75% which were used to produce the BC₂F₁ generation. BC₁-P₇ was the best BC₁F₁ plant because it had the highest recovery at 83.40% and was positive to Hsp-linked markers (Hsp70-u2 and AGi42). After three successive generations of backcrossing, the average genome recovery of the recurrent parent in the selected plants in BC₃F₁ was 95.37%. Hsp gene expression analysis was carried out on BC₁F₁, BC₂F₁ and BC₃F₁ selected lines. The Hsp genes were found to be up-regulated when exposed to heat treatment. The pattern of Hsp expression in the backcross generations was similar to that of the donor parent. This confirms the successful introgression of a stress-responsive gene (Hsp) into a Kulai chilli pepper variety. Furthermore, the yield performance viz. plant height, number of fruits, fruit length and weight and total yield of the improved plant were similar with the recurrent parent except that the plant height was significantly lower than the Kulai (recurrent) parent.     

Molecular markers for wheat leaf rust resistance gene Lr41

Leaf rust, caused by Puccinia triticina Eriks., is an important foliar disease of common wheat (Triticum aestivum L.) worldwide. Pyramiding several major rust-resistance genes into one adapted cultivar is one strategy for obtaining more durable resistance. Molecular markers linked to these genes are essential tools for gene pyramiding. The rust-resistance gene Lr41 from T. tauschii has been introgressed into chromosome 2D of several wheat cultivars that are currently under commercial production. To discover molecular markers closely linked to Lr41, a set of near-isogenic lines (NILs) of the hard winter wheat cultivar Century were developed through backcrossing. A population of 95 BC₃F_2:6 NILs were evaluated for leaf rust resistance at both seedling and adult plant stages and analyzed with simple sequence repeat (SSR) markers using bulked segregant analysis. Four markers closely linked to Lr41 were identified on chromosome 2DS; the closest marker, Xbarc124, was about 1 cM from Lr41. Physical mapping using Chinese Spring nullitetrasomic and ditelosomic genetic stocks confirmed that markers linked to Lr41 were on chromosome arm 2DS. Marker analysis in a diverse set of wheat germplasm indicated that primers BARC124, GWM210, and GDM35 amplified polymorphic bands between most resistant and susceptible accessions and can be used for marker-assisted selection in breeding programs.     

Selection strategies for marker-assisted backcrossing with high-throughput marker systems

Application of marker-assisted backcrossing for gene introgression is still limited by the high costs of marker analysis. High-throughput (HT) assays promise to reduce these costs, but new selection strategies are required for their efficient implementation in breeding programs. The objectives of our study were to investigate the properties of HT marker systems compared to single-marker (SM) assays, and to develop optimal selection strategies for marker-assisted backcrossing with HT assays. We employed computer simulations with a genetic model consisting of 10 chromosomes of 160 cM length to investigate the introgression of a dominant target gene. We found that a major advantage of HT marker systems is that they can provide linkage maps with equally spaced markers, whereas the possibility to provide linkage maps with high marker densities smaller than 10 cM is only of secondary use in marker-assisted backcrossing. A three-stage selection strategy that combines selection for recombinants at markers flanking the target gene with SM assays and genome-wide background selection with HT markers in the first backcross generation was more efficient than genome-wide background selection with HT markers alone. Selection strategies that combine SM and HT assays were more efficient than genome-wide background selection with HT assays alone. This result was obtained for a broad range of cost ratios of HT and SM assays. A further considerable reduction of the costs could be achieved if the population size in the first backcross generation was twice the population size in generations BC₂ and BC₃ of a three-generation backcrossing program. We conclude that selection strategies combining SM and HT assays have the potential to greatly increase the efficiency and flexibility of marker-assisted backcrossing.     

Identification of agronomically favorable quantitative trait loci alleles from a dent corn inbred Dan232 using advanced backcross QTL analysis and comparison with the F<Subscript>2:3</Subscript> population in popcorn

Specific traits are an important consideration in plant breeding. In popcorn, inferior agronomic traits could be improved using dent or flint corn backcrossed with popcorn. In this study, we used advanced backcross quantitative trait locus (AB-QTL) analysis to identify trait-improving QTL alleles from a dent maize inbred Dan232, and compared the detection of QTL in the BC₂S₁ population with QTL results using F_2:3 families of the same population. Two hundred and twenty BC₂S₁ families developed from a cross between Dan232 and an elite popcorn inbred N04 were evaluated for nine plant traits in replicated field trials under two environments. Using composite interval mapping (CIM), a total of 28 significant QTL were detected, and of these, 23 (82.14%) had favorable alleles contributed by the dent corn parent Dan232. Nine QTL (32.14%) detected in the BC₂S₁ population were also located in or near the same chromosome intervals in the F_2:3 population. All of the favorable QTL alleles from Dan232 could be used in marker-assisted selection (MAS) to improve the respective plant traits in popcorn breeding. In addition, their near isogenic lines (QTL-NILs) could be obtained through selfing or another 1–2 backcross with N04. Also, N04 improved for the studied plant traits could be developed from the BC₂S₁ families used in this study. This study demonstrated that the AB-QTL method can be applied to identify favorable QTL from dent corn inbred in popcorn breeding and, once identified, the alleles could be used in marker-assisted selection to improve the respective plant traits.     

Mapping and Marker-assisted Selection of a Brown Planthopper Resistance Gene bph2 in Rice (Oryza sativa L.)

Nilaparvata lugens Stål (brown planthopper, BPH), is one of the major insect pests of rice (Oryza sativa L.) in the temperate rice-growing region. In this study, ASD7 harboring a BPH resistance gene bph2 was crossed to a susceptible cultivar C418, a japonica restorer line. BPH resistance was evaluated using 134 F_2:3 lines derived from the cross between “ASD7” and “C418”. SSR assay and linkage analysis were carried out to detect bph2. As a result, the resistant gene bph2 in ASD7 was successfully mapped between RM7102 and RM463 on the long arm of chromosome 12, with distances of 7.6 cM and 7.2 cM, respectively. Meanwhile, both phenotypic selection and marker-assisted selection (MAS) were conducted in the BC₁F₁ and BC₂F₁ populations. Selection efficiencies of RM7102 and RM463 were determined to be 89.9% and 91.2%, respectively. It would be very beneficial for BPH resistance improvement by using MAS of this gene.     

Marker-assisted introgression of blackmold resistance QTL alleles from wild Lycopersicon cheesmanii to cultivated tomato (L. esculentum) and evaluation of QTL phenotypic effects

Blackmold, caused by the fungus Alternaria alternata, is a major ripe fruit disease of processing tomatoes. Previously, we found blackmold resistance in a wild tomato (Lycopersicon cheesmanii) and quantitative trait loci (QTL) for resistance were mapped in an interspecific population. Five QTLs were selected for introgression from L. cheesmanii into cultivated tomato using marker-assisted selection (MAS). Restriction fragment length polymorphism and PCR-based markers flanking, and within, the chromosomal regions containing QTLs were used for MAS during backcross and selfing generations. BC₁ plants heterozygous at the QTLs, and subsequent BC₁S₁ and BC₁S₂ lines possessing different homozygous combinations of alleles at the target QTLs, were identified using DNA markers. Field experiments were conducted in 1998 (with 80 marker-selected BC₁S₂ lines) and 1999 (with 151 marker-selected BC₁S₂ and BC₁S₃ lines) at three California locations. Blackmold resistance was assessed during both years, and horticultural traits were evaluated in 1999. The BC₁S₂ and BC₁S₃ lines containing L. cheesmanii alleles at the QTLs were associated with a large genetic variance for resistance to blackmold and moderate heritability, suggesting that significant genetic gain may be achieved by selection in this genetic material. L. cheesmanii alleles at three of the five introgressed QTLs showed a significant, positive effect on blackmold resistance. A QTL on chromosome 2 had the largest positive effect on blackmold resistance, alone and in combination with other QTLs, and was also associated with earliness, a positive horticultural trait. The other four QTLs were associated primarily with negative horticultural traits. Fine mapping QTLs using near isogenic lines could help determine if such trait associations are due to linkage drag or pleiotropy.     

Molecular marker-assisted backcrossing breeding: an example to transfer a thermostable β-amylase gene from wild barley

Molecular marker-assisted backcrossing (MABC) is widely recommended for transferring favorable alleles from a donor to an elite variety. The question remains whether MABC is an effective approach to developing a competitive commercial variety. Here, we illustrate the transfer of a thermostable β-amylase allele Sd3 from wild barley into a commercial barley variety Gairdner. The elite lines were chosen for the Regional Crop Variety Test that followed a standard conventional breeding process. The results demonstrated that the Sd3 allele not only increased enzyme thermostability but dramatically enhanced diastatic power, an important malting quality trait. The BC₁F₁ individuals had a fundamental impact on the comprehensive agronomic and quality traits of the final progenies, demonstrating the importance of screening at the early stage of backcrossing in MABC. There was sufficient genetic variation in the BC₃F₃ families to select other malting quality and agronomic traits. Ten individual breeding lines with improved β-amylase thermostability also had improved yields and grain plumpness. Three elite lines with improved malting quality and agronomic traits were selected to provide a parental line to incorporate the wild barley allele for breeding a commercial variety. A new strategy should be considered that uses marker-assisted selection and backcrossing to transfer a favorable allele from a wild parent.