Molecular Marker-assisted Backcrossing Research Articles

Phenotypical and molecular characterization of new pepper genotypes resistant to Chili pepper mild mottle virus firstly detected in Europe and other tobamoviruses

The tobamoviruses pose a recent threat to local varieties of Gernika pepper (cv. “Derio”) and Ibarra chili (cv. “Ibarroria”), causing a substantial decrease in both yield and fruit quality. Tobamoviruses such as Tomato mosaic virus (ToMV), Tobacco mild green mosaic virus (TMGMV) or Pepper mild mottle virus (PMMoV) are the viruses that cause the greatest impact in pepper crops of northern Spain. The emergence of a novel Tobamovirus, Chili pepper mild mottle virus (CPMMoV; GenBank MN164455.1), firstly identified in Europe, exacerbates these hazards. This situation has jeopardized the ongoing cultivation in some of the virus affected plots. A molecular marker-assisted backcrossing (MABC) breeding program was thus initiated to introduce the L3 and L4 resistance genes into these susceptible local varieties. MABC consisted of one crossing followed by four backcrosses and two self-fertilizations. After each crossbreeding event, the selection criteria of plants were based on plants resistance and fruits resemblance with the original sensitive varieties. This study allowed the homozygous introgression of resistance genes in the new obtained genotypes, reducing the reliance on hybrid seed production and associated costs. The resulting genotypes demonstrated robust resistance when inoculated with PMMoV 1.2, PMMoV 1.2.3, and CPMMoV. In addition, greenhouse yield trials were performed during two consecutive years to identify and select the most productive genotypes harboring the L3 or L4 gene, with the aim of registering some of them as commercial varieties. The productivity of the new selected genotypes did not display significant differences when compared to the original sensitive varieties. The introduction of genetic resistance in these local pepper varieties not only mitigates the immediate hazard posed by Tobamovirus but also fosters the preservation of local agricultural genetic resources, ensuring their continued cultivation and economic sustainability.

Stacking Multiple Genes Improves Resistance to Chilo suppressalis, Magnaporthe oryzae, and Nilaparvata lugens in Transgenic Rice.

The ability of various pests and diseases to adapt to a single plant resistance gene over time leads to loss of resistance in transgenic rice. Therefore, introduction of different pest and disease resistance genes is critical for successful cultivation of transgenic rice strains with broad-spectrum resistance to multiple pathogens. Here, we produced resistance rice lines with multiple, stacked resistance genes by stacking breeding and comprehensively evaluated their resistance to Chilo suppressalis (striped rice stemborer), Magnaporthe oryzae (rice blast), and Nilaparvata lugens (brown planthopper) in a pesticide-free environment. CRY1C and CRY2A are exogenous genes from Bacillus thuringiensis. Pib, Pikm, and Bph29 are natural genes in rice. CH121TJH was introduced into CRY 1C, Pib, Pikm, and Bph29. CH891TJH and R205XTJH were introduced into CRY 2A, Pib, Pikm, and Bph29. Compared with those observed in their recurrent parents, CH121TJH significantly increased the mortality of borers. The other two lines CH891TJH and R205XTJH are the same result. Three lines introduction of Pib and Pikm significantly reduced the area of rice blast lesions, and introduction of Bph29 significantly reduced seedling mortality from N. lugens. Introduction of the exogenous genes had relatively few effects on agronomic and yield traits of the original parents. These findings suggest that stacking of rice resistance genes through molecular marker-assisted backcross breeding can confer broad spectrum and multiple resistance in differently genetic backgrounds.

Improving Rice Blast Resistance by Mining Broad-Spectrum Resistance Genes at Pik Locus

Magnaporthe oryzae is known for its genetic diversity and pathogenic variability, leading to rapid breakdown of resistance in rice. Incorporating multiple broad-spectrum blast resistance genes into rice cultivars would extend disease resistance longevity. Effective resistance breeding in rice therefore requires continual enrichment of the reservoir of resistance genes and alleles. We conducted a large-scale screen of rice blast resistance in about 2 000 rice accessions. Among them, 247 accessions showed at least medium resistance to the natural infection of rice blast and 7 novel Pik alleles were identified from them. Variations in gene sequences were then correlated with the phenotypic trait to enable the identification of favorable alleles. Among the seven novel Pik alleles, the resistant rate of Pik-R0/ME/7017 donors was greater than 80%, and the disease score was less than 3. Through molecular marker-assisted backcross breeding, we successfully transferred the three Pik alleles, Pik-R0/ME/7017, into an elite cultivated line Kongyu 131 to obtain BC3F2 lines, which showed enhanced resistance to rice blast compared with the recurrent parent. Assessment of these near-isogenic lines in the greenhouse using 31 isolates of M.oryzae from Heilongjiang Province of China revealed that the resistant levels of the BC3F2 lines with Pik-R0/ME/7017 were significantly higher than those of the established cloned resistance genes Pik-m and Pi1. Exploring such alleles will enrich our gene library for resistance to rice blast.

Improvement of a Yairipok Chujak Maize Landrace from North Eastern Himalayan Region for β-Carotene Content through Molecular Marker-Assisted Backcross Breeding.

In the North Eastern Himalayan region (NEHR) of India, maize is an important food crop. The local people cultivate the maize landraces and consume them as food. However, these landraces are deficient in β-carotene content. Thus, we aimed to incorporate the crtRB1 gene from UMI285β+ into the genetic background of the NEHR maize landrace, Yairipok Chujak (CAUM66), and thereby enhance the β-carotene content through marker-assisted backcrossing (MABC). In this regard, we backcrossed and screened BC1F1 and BC2F1 plants possessing the heterozygous allele for crtRB1 and then screened with 106 polymorphic simple sequence repeat (SSR) markers. The plants having maximum recurrent parent genome recovery (RPGR) were selected in each generation and selfed to produce BC2F2 seeds. In the BC2F2 generation, four plants (CAUM66-54-9-12-2, CAUM66-54-9-12-11, CAUM66-54-9-12-13, and CAUM66-54-9-12-24) having homozygous crtRB1-favorable allele with maximum RPGR (86.74–90.16%) were selected and advanced to BC2F3. The four selected plants were selfed to produce BC2F3 and then evaluated for agronomic traits and β-carotene content. The agronomic performance of the four lines was similar (78.83–99.44%) to that of the recurrent parent, and β-carotene content (7.541–8.711 μg/g) was on par with the donor parent. Our study is the first to improve the β-carotene content in NEHR maize landrace through MABC. The newly developed lines could serve as potential resources to further develop nutrition-rich maize lines and could provide genetic stock for use in breeding programs.

Improving rice blast resistance of Feng39S through molecular marker-assisted backcrossing

BackgroundRice blast caused by Magnaporthe oryzae is one of the most widespread biotic constraints that threaten rice production. Using major resistance genes for rice blast resistance improvement is considered to be an efficient and technically feasible approach to achieve optimal grain yield.ResultsWe report here the introgression of the broad-spectrum blast resistance gene Pi2 into the genetic background of an elite PTGMS line, Feng39S, for enhancing it and its derived hybrid blast resistance through marker-assisted backcrossing (MABC) coupled with genomics-based background selection. Two PTGMS lines, designated as DB16206–34 and DB16206–38, stacking homozygous Pi2 were selected, and their genetic background had recurrent parent genome recovery of 99.67% detected by the SNP array RICE6K. DB16206–34 and DB16206–38 had high resistance frequency, with an average of 94.7%, when infected with 57 blast isolates over 2 years, and the resistance frequency of their derived hybrids ranged from 68.2% to 95.5% under inoculation of 22 blast isolates. The evaluation of results under natural blast epidemic field conditions showed that the selected PTGMS lines and their derived hybrids were resistant against leaf and neck blast. The characterizations of the critical temperature point of fertility-sterility alternation of the selected PTGMS lines, yield, main agronomic traits, and rice quality of the selected PTGMS lines and their hybrids were identical to those of the recurrent parent and its hybrids. DB16206–34/9311 or DB16206–38/9311 can be used as a blast-resistant version to replace the popular hybrid Fengliangyou 4. Likewise, DB16206–34/FXH No.1 or DB16206–38/FXH No.1 can also be used as a blast-resistant version to replace another popular hybrid Fengliangyou Xiang 1.ConclusionsOur evaluation is the first successful case to apply MABC with genomics-based background selection to improve the blast resistance of PTGMS lines for two-line hybrid rice breeding.

Transcriptome Dynamics of Double Recessive Mutant, o2o2o16o16, Reveals the Transcriptional Mechanisms in the Increase of Its Lysine and Tryptophan Content in Maize

In maize, pyramiding of o2 and o16 alleles can greatly improve the nutritional quality of grains. To dissect its molecular mechanism, we created a double recessive mutant line, o2o2o16o16, by introgression of the o2 and o16 alleles into the wild-type maize inbred line, by molecular marker-assisted backcross selection. The kernels (18 day after pollination (DAP), 28 DAP, and 38 DAP) of the o2o2o16o16 mutant and its parent lines were subject to RNA sequencing (RNA-Seq). The RNA-Seq analysis revealed that 59 differentially expressed genes (DEGs) were involved in lysine metabolism and 43 DEGs were involved in tryptophan metabolism. Among them, the genes encoding AK, ASADH, and Dap-F in the lysine synthesis pathway were upregulated at different stages of endosperm development, promoting the synthesis of lysine. Meanwhile, the genes encoding LKR/SDH and L-PO in the lysine degradation pathway were downregulated, inhibiting the degradation of lysine. Moreover, the genes encoding TAA and YUC in the tryptophan metabolic pathway were downregulated, restraining the degradation of tryptophan. Thus, pyramiding o2 and o16 alleles could increase the lysine and tryptophan content in maize. These above results would help to uncover the molecular mechanisms involved in the increase in lysine and the tryptophan content, through the introgression of o2 and o16 alleles into the wild-type maize.

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.

Marker-assisted introgression of resistance to fusarium wilt race 2 in Pusa 256, an elite cultivar of desi chickpea.

Fusarium wilt caused by F. oxysporum f. sp. ciceris causes extensive damage to chickpea (Cicer arietinum L.) in many parts of the world. In the central part of India, pathogen race 2 (Foc 2) causes severe yield losses. We initiated molecular marker-assisted backcrossing (MABC) using desi cultivar, Vijay, as a donor to introgress resistance to this race (Foc2) in Pusa 256, another elite desi cultivar of chickpea. To confirm introgression of resistance for this race, foreground selection was undertaken using two SSR markers (TA 37 and TA110), with background selection to observe the recovery of recurrent parent genome using 45 SSRs accommodated in 8 multiplexes. F1 plants were confirmed with molecular markers and backcrossed with Pusa 256, followed by cycles of foreground and background selection at each stage to generate 161 plants in BC3F2 during the period 2009-2013. Similarly, 46 BC3F1 plants were also generated in another set during the same period. On the basis of foreground selection, 46 plants were found homozygotes in BC3F2. Among them, 17 plants recorded >91% background recovery with the highest recovery percentage of 96%. In BC3F1 also, 14 hybrid plants recorded a background recovery of >85% with the highest background recovery percentage of >94%. The identified plants were selfed to obtain 1341 BC3F3 and 2198 BC3F2 seeds which were screened phenotypically for resistance to fusarium wilt (race 2) besides doing marker analysis. Finally, 17 BC3F4 and 11 BC3F3 lines were obtained which led to identification of 5 highly resistant lines of Pusa 256 with Foc 2 gene introgressed in them. Development of these lines will help in horizontal as well as vertical expansion of chickpea in central part of India.

Molecular Breeding of Rice Restorer Lines and Hybrids for Brown Planthopper (BPH) Resistance Using the Bph14 and Bph15 Genes.

BackgroundThe development of hybrid rice is a practical approach for increasing rice production. However, the brown planthopper (BPH), Nilaparvata lugens Stål, causes severe yield loss of rice (Oryza sativa L.) and can threaten food security. Therefore, breeding hybrid rice resistant to BPH is the most effective and economical strategy to maintain high and stable production. Fortunately, numerous BPH resistance genes have been identified, and abundant linkage markers are available for molecular marker-assisted selection (MAS) in breeding programs. Hence, we pyramided two BPH resistance genes, Bph14 and Bph15, into a susceptive CMS restorer line Huahui938 and its derived hybrids using MAS to improve the BPH resistance of hybrid rice.ResultsThree near-isogenic lines (NILs) with pyramided Bph14 and Bph15 were obtained by molecular marker-assisted backcross (MAB) and phenotypic selection. The genomic components of these NILs were detected using the whole-genome SNP (Single nucleotide polymorphism) array, RICE6K, suggesting that the recurrent parent genome (RPG) recovery of the NILs was 87.88, 87.70 and 86.62 %, respectively. BPH bioassays showed that the improved NILs and their derived hybrids carrying homozygous Bph14 and Bph15 were resistant to BPH. However, the hybrids with heterozygous Bph14 and Bph15 remained susceptible to BPH. The developed NILs showed no significant differences in major agronomic traits and rice qualities compared with the recurrent parent. Moreover, the improved hybrids derived from the NILs exhibited better agronomic performance and rice quality compared with the controls under natural field conditions.ConclusionsThis study demonstrates that it is essential to stack Bph14 and Bph15 into both the maternal and paternal parents for developing BPH-resistant hybrid rice varieties. The SNP array with abundant DNA markers is an efficient tool for analyzing the RPG recovery of progenies and can be used to monitor the donor segments in NILs, thus being extremely important for rice molecular breeding.

Introgression of null allele of Kunitz trypsin inhibitor through marker-assisted backcross breeding in soybean (Glycine max L. Merr.).

BackgroundPresence of Kunitz trypsin inhibitor (KTI) in soybean seeds necessitates pre-heat treatment of the soy-flour for its inactivation before using it in food and feed products. The heat treatment not only enhances processing costs of the soy-based foods and feeds but also affects seed-protein quality and solubility. Genetic elimination of KTI is an important and effective approach. Therefore, molecular marker-assisted backcross breeding (MABB) approach was adopted for genetic elimination of KTI from two popular soybean genotypes, DS9712 and DS9814. PI542044, an exotic germplasm line was used as donor of the kti allele which inhibits functional KTI peptide production.ResultsForeground selection for the kti allele was performed with three closely linked SSR markers while background selection was done with 93 polymorphic SSR markers. Plants in the BC1F1 generation were found to recover 70.4–87.63 % and 60.26–73.78 % of the recurrent parent genome (RPG) of DS9712 and DS9814, respectively. Similarly, selected plants in the BC2F1 generation had 93.01–98.92 % and 83.3–91.67 % recovery of their respective RPGs. Recombinant selection was performed so as to identify plants with minimal linkage drag. Biochemical analysis of the seeds of the selected plants (ktikti) confirmed absence of KTI peptides in the seeds. Phenotypically, the selected plants were comparable to the respective recurrent parent in yield and other traits.ConclusionsMABB approach helped in speedy development of 6 KTI free breeding lines of soybean. Such lines will be suitable for the farmers and the soybean industries to use in production of soy-based foods and feeds without pre-heat treatment of the soy-flour. It would contribute towards wider acceptability of soy-based foods and feeds.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0413-2) contains supplementary material, which is available to authorized users.

INTROGRESSION OF STRIGA RESISTANCE INTO POPULAR SUDANESE SORGHUM VARIETIES USING MARKER ASSISTED SELECTION

Witchweed (Strigas spp.) is one of the most important cereals production constraints globally and is projected to worsen with anticipated climate change. It is especially a devastating parasitic weed in Sub-Saharan Africa and parts of Asia. Integrated management strategies that depend mainly on host plant resistance provide the most effective control mechanism for Striga. We used molecular marker-assisted backcrossing to introgress Striga resistance from a resistant genotype, N13, into agronomically important genetic backgrounds (Tabat and Wad Ahmed). Backcross populations BC3S3 were generated and genotyped using Simple Sequence Repeat (SSR) and Diversity Arrays Technology (DArT) markers. A total of 17 promising backcross progenies were selected and screened in Striga infested field alongside their parents. The Area Under Striga Progress Curve (AUSPC) showed significant decrease in Striga count (920-7.5) resulting in a 97-189% increase in yield under Striga pressure. Our results demonstrate the practical application of marker assisted selection (MAS) to generate farmer-preferrd Striga resistant lines in Sudan.

Evaluation on Effect of Resistance Improvement of Bacterial Leaf Blight and Brown Planthopper for Three Dominant Indica Restorer Lines

A novel bacterial blight (BB) resistance gene, Xa23, and brown planthopper (BPH) resistance gene, Bph18(t) were introgressed into three popular restorer lines (Minghui 86, Shuhui 527, and Zhehui 7954) for wild abortive cytoplasmic male sterility by marker-assisted backcross and pyramiding breeding approaches in combination with artificial inoculation and stringent phenotypic selections. The four derived BB resistant restorer lines [Minghui 86-Xa23, Shuhui 527-Xa23, Zhehui 7954-Xa23, and Zheshu-Xa23-Bph18(t)] and their derived hybrid combinations with Ⅱ-32A and Huhan11A demonstrated similar BB resistance spectrum as the donor parent, IRBB23. The three derived BPH resistant restorer lines [Shu 527-Bph18(t), Zhehui 7954-Bph18(t), and Zheshu-Xa23-Bph18(t)] showed resistant or moderate resistant to BPH. The newly developed BB and BPH resistant restorers and their derived hybrids were identical to their respective original versions for agronomic traits especially under disease free condition. However, under severe disease condition, the four BB resistant restorer lines exhibited significantly higher grain weight and spikelet fertility as compared to the respective original restorer lines thus further resulting in BB resistant hybrids with significantly higher grain yields than their respective original hybrids. The results indicated that the Xa23 gene could completely express its dominant resistance in different backgrounds of both restorer and male sterile lines across different growth stages,suggesting its immense breeding value in BB resistance improvement for hybrid rice. However, effect of resistant improvement of Bph18(t) for BPH depends on genetic background. Improvement of resistance of BB and BPH for restorer lines by molecular marker-assisted backcross and pyramiding was deeply discussed.

Introgression of bacterial blight (BB) resistance genes Xa7 and Xa21 into popular restorer line and their hybrids by molecular marker-assisted backcross (MABC) selection scheme

Yihui1577 is an elite restorer line widely used in hybrid rice production in China, however, both the restorer and their derived hybrids are susceptible to bacterial blight (BB) caused by Xathomonas oryzae pv. oryzae ( Xoo ). In order to overcome this problem, we had introgressed two resistant genes Xa7 and Xa21 into Yihui1577 by marker-assisted backcross (MABC) with foreground selection scheme to speed up the process. Six breeding lines with different BB resistance genes: HH1202 ( Xa7 ), HH1203 ( Xa7 ), HH1204 ( Xa21 ), HH1205 ( Xa21 ), HH1206 ( Xa7+Xa21 ) and HH1207 ( Xa7+Xa21 ) were selected and crossed with four CMS and one TGMS lines. Seven most virulent and prevalent Xoo strains (PXO61, PXO99, ZHE173, GD1358, FuJ, YN24 and HeN11) from the Philippines and different provinces of China were inoculated for evaluating the BB-resistance of the selected lines and their derived hybrids. The results reveal that the two lines and their derived hybrids with single resistance gene Xa7 were resistant against six of the seven Xoo strains, except for PXO99. The lines with single resistance gene Xa21 were only susceptible to the Xoo strain FuJ, but some of their derived hybrids were susceptible to the Xoo strains FuJ and GD1358. Interestingly, the pyramiding lines carrying the two resistance genes Xa7 and Xa21 and also their derived hybrids were resistant against all the seven Xoo strains. The data of agronomic and grain quality characteristics demonstrated that the selected lines were similar to that of the recurrent parent Yihui1577. Corrective measures taken by way of introgression of BB-resistance genes: Xa7 and Xa21 into the popular restorer line, Yihui1577 through MABC approach for enhancing the BB-resistance level was effective and timely. Keywords: Bacterial blight, resistance gene, Xa7 and Xa21 , MABC, inoculation and reaction, agronomic traits, grain quality

Transfer of resistance to potato virus�Y (PVY) from Nicotiana africana to Nicotiana tabacum: possible influence of tissue culture on the rate of introgression

A disomic chromosome addition line of tobacco, Nicotiana tabacum L., was established previously that possesses a single chromosome pair from N. africana [Merxm. and Buttler]. This addition chromosome carries a gene that confers increased resistance to severe strains of potato virus Y (PVY). Methods to increase the probability of gene transfer from alien chromosomes to tobacco (2n=48) are desired. In the research described here, the PVY resistance gene was transferred to a tobacco chromosome from the N. africana addition chromosome in seven independent cases. One introgression event was obtained using conventional backcrossing of the disomic addition line to N. tabacum cv. Petite Havana, while the remaining six events were obtained using a scheme that involved exposure of explants of the addition line to tissue culture. Twenty-six derived 2n=48 individuals heterozygous for PVY resistance were found to exhibit 24 bivalents or 23 bivalents + 2 univalents at metaphase I. Ovular transmission rates for the PVY resistance factor ranged from 25% to 52%, while pollen transmission rates were much lower, ranging from 0 to 39%. Fifty-one random amplified polymorphic DNA (RAPD) markers specific for the intact addition chromosome were identified and used to characterize derived 2n=48/PVY-resistant genotypes. Variability was observed among these plants with respect to the total number of N. africana RAPD markers that were present, which is an indication that crossing over was occurring within each of the seven introgressed chromosome segments. A limited molecular marker-assisted backcrossing experiment allowed for selection of a 2n=48/PVY-resistant individual that possessed only 6 of the 51 original N. africana RAPD markers. In vitro culture is potentially a valuable system for increasing the rate of alien gene transfer in tobacco, and the successful transfer of PVY resistance from N. africana may allow for an increased level and range of resistance to this virus in tobacco.

Fine mapping of a malting-quality QTL complex near the chromosome 4H S telomere in barley.

Malting quality has long been an active objective in barley (Hordeum vulgare L.) breeding programs.However, it is difficult for breeders to manipulate malting-quality traits because of inheritance complexity and difficulty in evaluation of these quantitative traits. Quantitative trait locus (QTL) mapping provides breeders a promising basis with which to manipulate quantitative trait genes. A malting-quality QTL complex, QTL2, was mapped previously to a 30-cM interval in the short-arm telomere region of barley chromosome 4H in a "Step-toe"/"Morex" doubled haploid population by the North American Barley Genome Project, using an interval mapping method with a relatively low-resolution genetic map. The QTL2 complex has moderate effects on several malting-quality traits, including malt extract percentage(ME), a-amylase activity (AA), diastatic power (DP), malt 13-glucan content (BG), and seed dormancy, which makes it a promising candidate gene source in malting barley-cultivar development. Fine mapping QTL2 is desirable for precisely studying barley malting-quality trait inheritance and for efficiently manipulating QTL2 in breeding. A reciprocal-substitution mapping method was employed to fine map QTL2. Molecular marker-assisted backcrossing was used to facilitate the generation of isolines. Fourteen different types of "Steptoe" isolines, including regenerated "Steptoe" and 13 different types of "Morex" isolines,including regenerated "Morex", were made within a 41.5-cM interval between MWG634 and BCD265B on chromosome 4H. Duplicates were identified for 12 "Steptoe" and 12 "Morex" isoline types. The isolines together with "Steptoe" and "Morex" were grown variously at three locations in 2 years for a total of five field environments.Four malting-quality traits were measured: ME, DP, AA,and BG. Few significant differences were found between duplicate isolines for these traits. A total of 15 putative QTLs were mapped; three for ME, four for DP, six for AA,and two for BG. Background genotype seemed to make a difference in expression/detection of QTLs. Of the 15 QTLs identified, ten were from the "Morex" and only five from the "Steptoe" background. By combining the results from different years, field environments, and genetic backgrounds and taking into account overlapping QTLsegments, six QTLs can be conservatively estimated: two each for ME and AA and one each for DP and BG with chromosome segments ranging from 0.7 cM to 27.9 cM. A segment of 15.8 cM from the telomere (MWG634-CDO669) includes all or a portion of all QTLs identified. Further study and marker-assisted breeding should focus on this 15.8-cM chromosome region.

Dissection of a malting quality QTL region on chromosome 1 (7H) of barley

Malting and brewing are major uses of barley (Hordeum vulgare L.) worldwide, utilizing 30–40% of the crop each year. A set of complex traits determines the quality of malted barley and its subsequent use for beer. Molecular genetics technology has increased our understanding of genetic control of the many malting and brewing quality traits, most of which are quantitatively inherited. The objective of this study was to further dissect and evaluate a known major malting quality quantitative trait locus (QTL) region of about 28 cM on chromosome 1 (7H). Molecular marker-assisted backcrossing was used to develop 39 isolines originating from a ‘Steptoe’ / ‘Morex’ cross. Morex, a 6–row malting type, was the donor parent and Steptoe, a 6–row feed type, was the recurrent parent. The isolines and parents were grown in four environments, and the grain was micro-malted and analyzed for malting quality traits. The effect of each Morex chromosome segment in the QTL target region was determined by composite interval mapping (CIM) and confirmed and refined by multiple interval mapping (MIM). One QTL was resolved for malt extract content, and two QTLs each were resolved for α-amylase activity, diastatic power, and malt β-glucan content. One additional putative malt extract QTL was detected at the plus border of the target region by CIM, but not confirmed by MIM. All QTLs were resolved to intervals of 2.0 to 6.4 cM by CIM, and to intervals of 2.0 cM or less by MIM. These results should facilitate marker-assisted selection in breeding improved malting barley cultivars.

Molecular marker-assisted selection for enhanced yield in malting barley

Brewers are reluctant to change malting barley (Hordeum vulgare ssp. vulgare L.) cultivars due to concerns of altered flavor and brewing procedures. The U.S. Pacific Northwest is capable of producing high yielding, high quality malting barley but lacks adapted cultivars with desirable malting characteristics. Our goal was to develop high yielding near isogenic lines that maintain traditional malting quality characteristics by transferring quantitative trait loci (QTL) associated with yield, via molecular marker-assisted backcrossing, from the high yielding cv. Baronesse to the North American two-row malting barley industry standard cv. Harrington. For transfer, we targeted Baronesse chromosome 2HL and 3HL fragments presumed to contain QTL that affect yield. Analysis of genotype and yield data suggests that QTL reside at two regions, one on 2HL (ABG461C-MWG699) and one on 3HL (MWG571A-MWG961). Genotype and yield data indicate that additional Baronesse genome regions are probably involved, but need to be more precisely defined. Based on yield trials conducted over 22 environments and malting analyses from 6 environments, we selected one isogenic line (00-170) that has consistently produced yields equal to Baronesse while maintaining a Harrington-like malting quality profile. We conclude there is sufficient data to warrant experiments testing whether the 2HL and 3HL Baronesse QTL would be effective in increasing the yield of other low yielding barley cultivars.

Genetic basis and mapping of the resistance to Rice yellow mottle virus. III. Analysis of QTL efficiency in introgressed progenies confirmed the hypothesis of complementary epistasis between two resistance QTLs

Our previous studies have hypothesised that a complementary epistasis between a QTL located on chromosome 12 and a QTL located on chromosome 7 was one of the major genetic factors controlling partial resistance to Rice yellow mottle virus (RYMV). We report research undertaken to verify this hypothesis and to introgress the resistant allele of these two QTLs from an upland resistant japonica variety, Azucena, into a lowland susceptible indica variety IR64. Three cycles of molecular marker-assisted back cross breeding were performed using RFLP and microsatellite markers. Resistance to RYMV was evaluated in F2 and F3 offspring of the BC1 and BC2 generations. Marker-assisted introgression (MAI) was very efficient: in the selected BC3 progeny the proportion of the recipient genome was close to 95% for the ten non-carrier chromosomes, and the length of the donor chromosome segment surrounding the two QTLs was less than 20 cM. The relevancy of the complementary epistasis genetic model proposed previously was confirmed experimentally: in BC1 and BC2 generations only F3 lines having the allele of the resistant parent on QTL12 and QTL7 show partial resistance to RYMV. Comparison of our experimental process of MAI with the recommendations of analytic and simulation studies pointed out the methodological flexibility of MAI. Our results also confirmed the widely admitted, but rarely verified, assumption that QTL-alleles detected in segregating populations could be treated as units of Mendelian inheritance and that the incorporation of these alleles into elite lines would result in an enhanced performance. The next step will be the design of tools for the routine use of molecular markers in breeding for partial resistance to RYMV and the development of material for the analysis of resistance mechanisms and the structure of a virus resistance gene in rice.

Inheritance of Anthracnose Resistance in Common Bean Differential Cultivar AB 136.

Inheritance of anthracnose resistance of the common bean (Phaseolus vulgaris L.) differential cultivar AB 136 to races 89, 64, and 73 (binary system designation) was studied in crosses with the susceptible differential cultivars Michelite (race 89), Mexico 222 (race 64), and Cornell 49-242 (race 73). In each cross two progenitors, the F1, F2, and backcross-derived plants were inoculated with the respective race under environmentally controlled greenhouse conditions. The results indicate that single dominant gene(s) control resistance to races 89 and 64, giving a segregation ratio of 3:1 in the F2, 1:0 in the backcrosses to AB 136, and 1:1 in the backcross to Michelite (race 89), and to Mexico 222 (race 64). For race 73, the following segregation ratios between resistant and susceptible plants were observed: 13:3 in the F2, 1:0 in the backcross to AB 136, and 1:1 in the backcross to Cornell 49-242. Such results suggest that two independent genes may determine resistance of AB 136 to race 73, one dominant (Co-6) and one recessive that is proposed to be assigned co-8. Genotypes Co-6_ or co-8 co-8 would condition resistance, whereas susceptibility would be present in genotypes co-6 co-6 Co-8_. Given the dominant nature of anthracnose resistance genes present in line AB 136 and its resistance to 25 races of Colletotrichum lindemuthianum identified in Brazil by other researchers, we included this cultivar as one of the donor parents in our molecular marker-assisted backcross breeding program, to develop common bean cultivars resistant to anthracnose and adapted to Central Brazil.

Mapping genes for resistance to barley stripe rust (Puccinia striiformis f. sp. hordei)

Two genes conferring resistance to the barley stripe rust found in Mexico and South America, previously identified as race 24, were mapped to the M arms of barley chromosomes 7 and 4 in a doubled haploid population using molecular markers and the quantitative trait loci (QTL) mapping approach. The resistance gene on chromosome 7 had a major effect, accounting for 57% of the variation in disease severity. The resistance gene on chromosome 4 had a minor effect, accounting for 10% of the variation in trait expression. Two pairs of restriction fragment length polymorphism markers are being used to introgress the resistance genes to North American spring barley using molecular marker-assisted backcrossing.