Testcross Progeny Research Articles

Accuracy of genomic selection in European maize elite breeding populations

Genomic selection is a promising breeding strategy for rapid improvement of complex traits. The objective of our study was to investigate the prediction accuracy of genomic breeding values through cross validation. The study was based on experimental data of six segregating populations from a half-diallel mating design with 788 testcross progenies from an elite maize breeding program. The plants were intensively phenotyped in multi-location field trials and fingerprinted with 960 SNP markers. We used random regression best linear unbiased prediction in combination with fivefold cross validation. The prediction accuracy across populations was higher for grain moisture (0.90) than for grain yield (0.58). The accuracy of genomic selection realized for grain yield corresponds to the precision of phenotyping at unreplicated field trials in 3-4 locations. As for maize up to three generations are feasible per year, selection gain per unit time is high and, consequently, genomic selection holds great promise for maize breeding programs.

Proposal for Shift to Reciprocal Recurrent Selections in “Clone and Strain Synthesis” Timothy Breeding using Molecular Marker Diversity

ABSTRACTIntegration of reciprocal recurrent selection (RRS) with the formation and use of heterotic groups is a key strategy for yield improvement in timothy (Phleum pratense L.) breeding. This study aims at utilizing putative heterotic groups based on molecular marker diversity in “clone and strain synthesis” (CSS) development. Strains are synthesized by combining two top‐cross progenies, that is, comprising two seed parental clones (SPs) and a pollen parental strain (PP). A significant positive correlation (r = 0.64, p < 0.05) was found between forage yields of 15 (= 6C2) CSS strains and genetic distance estimates among all possible pairs of their six SPs using 28 simple sequence repeat markers. This result suggests that the adoption of elite clones as SPs of CSS strains, selected from putative heterotic groups formed on the basis of molecular markers, results in the efficient development of high‐yielding strains with exploiting specific combining ability (SCA) effects among SPs. The use of CSS has some advantages compared with conventional cultivar synthesis. One is that RRS with two or three heterotic groups can be established in CSS. Another is that current leading cultivars can be utilized as the PP. These advantages and the results presented in this study has led us to a procedure that consists of (i) a division of germplasm pools based on molecular marker diversity and (ii) a confirmation of the separated groups based on test cross progeny tests. The procedure provides an effective introduction of RSS into CSS development.

Quantitative genetic parameters for selection of biomass yield in hybrid rye

With 2 figure and 6 tablesAbstractWinter rye is an attractive crop as substrate for biogas production. We aimed to estimate quantitative genetic parameters for biomass yield in elite hybrid materials. About 380 testcross progeny incorporating intrapool crosses and interpool hybrids were analysed in six experiments across each of three to four locations. Mean values of biomass yield on a 100% dry matter basis ranged between 152 and 160 dt/ha, and the best entries had 175 dt/ha across locations. Genotypic variances for biomass yield were significant (P < 0.01) in all experiments being higher for interpool hybrids. Genotype × location interaction variances were significant and, in most instances, of similar size than genotypic variances. Estimates of entry‐mean heritability were moderate. Correlations among traits were highly varying with experiments. Consistently, significant genotypic correlations with biomass yield were found for plant height (0.4–0.9) and early growth (0.2–0.8). In conclusion, broad genetic variation for biomass yield already existing in elite hybrid materials should stimulate selection for this newly adopted trait.

English

Information on the genetics of fertility restoration facilitates breeding and/or selection of restorer lines used in hybrid breeding program in a cytoplasmic male sterility (CMS) system. Inheritance of fertility restoration of WA type CMS in rice, (Oryza sativa L.) was studied utilizing IR58025A, IR62829A and IR68899A CMS lines and three restorers viz., Amol-2, IR50 and Poya. The F1s showed pollen and spikelet fertility similar to the restorer parents, indicating that restoration ability were dominant and the cytoplasmic-genetic sterility system of CMS lines were sporophytic in nature. Evaluation of fertility in F2 populations and testcross progenies (BC1) revealed that fertility restoration in Amol-2 and IR50 were controlled by two major dominant genes whereas, it was controlled by one dominant gene in Poya. Segregation for spikelet fertility in F2 and backcross generation conformed to the results on pollen fertility. Analysis of line x tester indicated pre-dominance of non-additive genetic variance. It suggested greater importance of non-additive gene action in pollen and spikelet fertility expression and indicated very good prospect for the exploitation of non-additive genetic variation for pollen and spikelet fertility through hybrid breeding.   Key words: Rice, genetics, male fertility restoration, combining ability.

Correlations and QTL detection in maize family per se and testcross progenies for plant height and ear height

With 2 figures and 4 tablesAbstractThe magnitude of genotypic correlations between family per se and testcross, r^g (per se, TC), is crucial for testing schemes in hybrid breeding as well as improvement of commercial hybrids. The objectives of this study were to identify the r^g (per se, TC), to examine the proportion of common QTL for both types of progenies and to determine the gene action of QTL identified for family per se and their value for the prediction of testcross progenies. Under multi‐environments, r^g (per se, TC) were significant and higher than r^p (per se, TC), the phenotypic correlations between family per se and testcross for plant height and ear height in two maize crosses. The final fit for QTL explained 21.6–70.3% phenotypic variance and 24.4–79.4% genotypic variance for family per se. However, in cross‐validation analysis (CV), the genotypic variance explained by all QTL was less than 60% for the two traits. Several common QTL (Chr. 1, Chr. 8 and Chr. 10) could be detected for both family per se and TC, which seems feasible to apply marker‐assisted selection in maize breeding programmes.

Association mapping in an elite maize breeding population

Association mapping (AM) is a powerful approach to dissect the genetic architecture of quantitative traits. The main goal of our study was to empirically compare several statistical methods of AM using data of an elite maize breeding program with respect to QTL detection power and possibility to correct for population stratification. These models were based on the inclusion of cofactors (Model A), cofactors and population effect (Model B), and SNP effects nested within populations (Model C). A total of 930 testcross progenies of an elite maize breeding population were field-evaluated for grain yield and grain moisture in multi-location trials and fingerprinted with 425 SNP markers. For grain yield, population stratification was effectively controlled by Model A. For grain moisture with a high ratio of variance among versus within populations, Model B should be applied in order to avoid potential false positives. Model C revealed large differences among allele substitution effects for trait-associated SNPs across multiple plant breeding populations. This heterogeneous SNP allele substitution effects have a severe impact for genomic selection studies, where SNP effects are often assumed to be independent of the genetic background.

Mapping of QTL for resistance to first and second generation of European corn borer using an integrated SNP and SSR linkage map

The European corn borer (ECB, Ostrinia nubilalis Hubner) is a major pest of maize (Zea mays L.) in North America and Europe. In this study, we mapped and characterized QTL affecting the resistance to the first (ECB1) and second generation (ECB2) of ECB and plant height with the aid of 164 SNP and 88 SSR markers. A total of 144 testcross (TC) progenies of double haploid (DH) lines developed from a cross of two parental lines from the Stiff Stalk germplasm pool were tested at six different locations in the USA under both natural and artificial infestation with ECB larvae. Resistance to ECB1 and ECB2 was assessed by measuring leaf feeding and stalk breakage, respectively, using a visual rating scale from 1 (severe damage) to 9 (no damage). Genotypic variance among the TC progenies was significant for all traits. Heritabilities were moderately high (0.69) for stalk breakage and plant height (0.75), but only moderate for leaf feeding (0.43). For stalk breakage, three QTL were detected that together explained 36% of the genetic variance, whereas for leaf feeding only one QTL was mapped, which explained 25% of the genotypic variance. For plant height, two QTL were identified, explaining 20% of the genotypic variance. The QTL for leaf feeding and stalk breakage mapped by us were located in chromosomal regions adjacent to those reported for other maize germplasm and therefore might be suitable candidates for marker-assisted selection (MAS) during line development.

Genome-based prediction of testcross values in maize

This is the first large-scale experimental study on genome-based prediction of testcross values in an advanced cycle breeding population of maize. The study comprised testcross progenies of 1,380 doubled haploid lines of maize derived from 36 crosses and phenotyped for grain yield and grain dry matter content in seven locations. The lines were genotyped with 1,152 single nucleotide polymorphism markers. Pedigree data were available for three generations. We used best linear unbiased prediction and stratified cross-validation to evaluate the performance of prediction models differing in the modeling of relatedness between inbred lines and in the calculation of genome-based coefficients of similarity. The choice of similarity coefficient did not affect prediction accuracies. Models including genomic information yielded significantly higher prediction accuracies than the model based on pedigree information alone. Average prediction accuracies based on genomic data were high even for a complex trait like grain yield (0.72-0.74) when the cross-validation scheme allowed for a high degree of relatedness between the estimation and the test set. When predictions were performed across distantly related families, prediction accuracies decreased significantly (0.47-0.48). Prediction accuracies decreased with decreasing sample size but were still high when the population size was halved (0.67-0.69). The results from this study are encouraging with respect to genome-based prediction of the genetic value of untested lines in advanced cycle breeding populations and the implementation of genomic selection in the breeding process.

The clf2 gene has an epigenetic role in the multifactorial etiology of cleft lip and palate in the A/WySn mouse strain

The A/WySn mouse strain with 15 to 20% penetrance of cleft lip and palate (CLP) is an animal model for human multifactorial CLP. The CLP is due to two unlinked genes that interact epistatically, Wnt9b(clf1) and clf2, plus a maternal effect. The Wnt9b(clf1) mutation is an IAP transposon insertion. The clf2 gene, with unknown function, was located in a 13.6 Mb region of chromosome 13 containing 145 genes. To reduce the clf2 candidate region, 1146 mice segregating for A/WySn and C57BL/6J alleles at clf2 were screened for recombinants by simple sequence-length polymorphism haplotypes; recombinants' testcross progeny were typed for CLP and simple-sequence length polymorphisms. To identify the function of clf2, the effect of clf2 genotype on risk of CLP was tested in Wnt9b(null/null) knockouts and in compound mutants (Wnt9b(clf1/null) ), and the methylation of the IAP at Wnt9b was assayed in the Wnt9b(clf1/null) mutants by combined bisulfite restriction analysis. The location of clf2 was redefined to 3.0 Mb between Cntnap3 and AK029746 containing 48 genes, of which 30 are Zfp genes. The clf2 genotype had no detectable effect on Wnt9b(null/null) embryos, but strongly affected risk of CLP and methylation of the IAP in Wnt9b(clf1/null) embryos. CLP was associated with low levels of methylation of the IAP. The clf2 gene is the first identified polymorphism that affects the epigenetic methylation and silencing of IAP retrotransposons. This CLP model raises the question of whether parallel epigenetic factors are involved in risk and environmental sensitivity of human CLP.

Correlation between per se and Testcross Performance in Rye (Secale cereale L.) Introgression Lines Estimated with a Bivariate Mixed Linear Model

ABSTRACTIn hybrid breeding a high correlation between per se performance of parental lines and hybrid performance can help to reduce the efforts for testcross trials. Our objectives were (i) to investigate the correlation between per se and testcross performance of rye (Secale cereale L.) candidate introgression lines (pre‐ILs) for thousand‐kernel weight, test weight, falling number, starch, pentosan, and protein content; and (ii) to investigate the consistency of quantitative trait loci (QTL) determined in pre‐ILs and their testcross progenies. Eighty pre‐ILs carrying donor chromosome segments (DCS) from the Iranian primitive rye Altevogt 14160 in the genetic background of elite line L2053‐N, were evaluated at four locations over 2 yr. Genetic correlations between per se and testcross performance were estimated with a bivariate mixed linear model. Putative QTL regions were detected by comparing the performance of pre‐ILs carrying certain DCS with the performance of the recurrent parent. Genotypic variances and heritabilities were higher for per se than for testcross performance. Genetic correlations between per se and testcross performance were mostly intermediate. Common QTL regions were observed for test weight, starch, pentosan, and protein content. Our results revealed that a superior per se performance of rye pre‐ILs is not indicative for a superior hybrid performance. Consequently, using rye pre‐ILs in hybrid breeding programs requires testcross trials.

Genetic Analysis of Cell Wall Traits Relevant to Cellulosic Ethanol Production in Maize (Zea mays L.)

Maize hybrids with improved stover yield and quality are needed to support the emerging cellulosic biofuels industry. Knowledge of the genetic variation, covariation, and genetic architecture of agronomic and cell wall traits will help maize breeders improve relevant traits through selective breeding and genetic engineering. We utilized the maize intermated B73 × Mo17 (IBM) recombinant inbred line (RIL) population to map quantitative trait loci (QTL) and to evaluate the genetic relationships among traits relevant for cellulosic biofuel production, including grain yield, stover yield, neutral detergent fiber digestibility (NDFD), and glucan, xylan, and lignin concentration. Intermated B73 × Mo17 RIL were evaluated per se and as testcrosses to W604S to assess how well performance and QTL effects correspond between inbreds and hybrids. There were no unfavorable correlations between agronomic and cell wall traits within the testcross progeny. Polysaccharide content was either positively correlated to, or not correlated to, NDFD, indicating that increasing both carbohydrate content and convertibility is feasible. Quantitative trait loci were identified for all traits evaluated, but few QTL were in common between inbred per se and testcross evaluations despite moderate to high genotypic correlations between progeny types. This study provides information that will support the development of dual‐purpose maize hybrids that are high in grain yield and also high in yield of digestible cellulosic feedstock.

The Dominant Ms Allele in Onion Shows Reduced Penetrance

The most commonly used source of cytoplasmic male sterility in onion (Allium cepa) is controlled by the interaction of the cytoplasm [male-sterile (S) or normal male-fertile (N)] and one nuclear male-fertility-restoration locus (Ms). Scoring of genotypes at Ms is generally done by testcrossing male-fertile to male-sterile (S msms) plants, followed by scoring of testcross progenies for male-fertility restoration. We identified two N-cytoplasmic families, one that was homozygous dominant and the other segregating at Ms. Plants from each of these two families were individually testcrossed to male-sterile onion. Nuclear restoration of male fertility in testcross progenies was evaluated in the field over 4 years. For male plants homozygous dominant at Ms, we expected testcross families to show 100% male-fertility restoration, but observed mean values between 46% and 100%. For plants segregating at Ms, we again observed lower than expected frequencies of male-fertility restoration. These results demonstrate that the dominant Ms allele shows reduced penetrance, requiring that male-fertility restoration be scored over years to more confidently assign genotypes at Ms.

Powdery Mildew Resistance in the Peach Cultivar Pamirskij 5 Is Genetically Linked with the Gr Gene for Leaf Color

Peach powdery mildew (Podosphaera pannosa var. persicae) is a major disease of peach trees [Prunus persica (L.) Batsch]. Various studies on powdery mildew resistance have been conducted in peach. The present study was initiated to determine the inheritance of powdery mildew resistance found in the green leaf peach rootstock ‘Pamirskij 5’. Crosses were carried out between ‘Pamirskij 5’ and the susceptible red leaf peach rootstock ‘Rubira’®. Segregation analysis performed in F1 (1:0), F2 (3:1), and test cross (1:1) progenies indicated single dominant gene control of peach powdery mildew resistance in ‘Pamirskij 5’. Cosegregation analysis provided clear evidence that the loci for powdery mildew resistance and the leaf color trait are very closely linked in ‘Pamirskij 5’. The estimated distance between these loci was 0 ± 3.26 cM for the F2 and 0 ± 0.00 cM for the test cross populations.

Segregation distortion in common wheat of a segment of Thinopyrum intermedium chromosome 7E carrying Bdv3 and development of a Bdv3 marker

AbstractYellow dwarf (YD) disease is one of the most destructive diseases of cereals worldwide. Wheat (Triticum aestivum L.)–Thinopyrum intermedium 7E(7D) substitution line P29 carries resistance to YD, known as Bdv3, that originates on the long arm of chromosome 7E of Th. intermedium, and the resistance was introgressed into wheat chromosome 7D as T7DS.7DL–7EL in the translocation lines P961341 and P98134. Until now, quantification of YD viruses in cereal crops was usually done by enzyme‐linked immunosorbent assay (ELISA), which is time consuming and laborious. To facilitate this analysis, SSR‐Bdv3, a diagnostic molecular marker, was developed in this study. The transmission of the Th. intermedium segment with Bdv3 was investigated using the SSR‐Bdv3 marker and ELISA in F2 and testcross progeny derived by crossing two wheat–Th. intermedium translocation lines to four common wheat cultivars. A Th. intermedium chromosome 7E segment in the translocation line P98134 was preferentially transmitted through male gametes in all of its crosses with the four wheat cultivars. However, the transmission frequency of the Th. intermedium 7E segment in another wheat–Th. intermedium translocation line, P961341, varied in different genetic backgrounds. The F2 populations from reciprocal crosses of Chinese Spring and P961341 showed good fits to the expected ratio of 1 : 2 : 1. In this study, male preferential transmission for either chromosome 7E or chromosome 7D was observed in the progeny derived by crossing P961341 to other wheat cultivars.

Hybrid maize breeding with doubled haploids: V. Selection strategies for testcross performance with variable sizes of crosses and S1 families

In hybrid maize (Zea mays L.) breeding, doubled haploids (DH) are increasingly replacing inbreds developed by recurrent selfing. Doubled haploids may be developed directly from S(0) plants in the parental cross or via S(1) families. In both these breeding schemes, we examined 2 two-stage selecting strategies, i.e., considering or ignoring cross and family structure while selection among and within parental crosses and S(1) families. We examined the optimum allocation of resources to maximize the selection gain DeltaG and the probability P(q) of identifying the q% best genotypes. Our specific objectives were to (1) determine the optimum number and size of crosses and S(1) families, as well as the optimum number of test environments and (2) identify the superior selection strategy. Selection was based on the evaluation of testcross progenies of (1) DH lines in both stages (DHTC) and (2) S(1) families in the first stage and of DH lines within S(1) families in the second stage (S(1)TC-DHTC) with uniform and variable sizes of crosses and S(1) families. We developed and employed simulation programs for selection with variable sizes of crosses and S(1) families within crosses. The breeding schemes and selection strategies showed similar relative efficiency for both optimization criteria DeltaG and P (0.1%). As compared with DHTC, S(1)TC-DHTC had larger DeltaG and P (0.1%), but a higher standard deviation of DeltaG. The superiority of S(1)TC-DHTC was increased when the selection was done among all DH lines ignoring their cross and family structure and using variable sizes of crosses and S(1) families. In DHTC, the best selection strategy was to ignore cross structures and use uniform size of crosses.

Identification of heterotic metabolite QTL in Arabidopsis thaliana RIL and IL populations

Two mapping populations of a cross between the Arabidopsis thaliana accessions Col-0 and C24 were cultivated and analyzed with respect to the levels of 181 metabolites to elucidate the biological phenomenon of heterosis at the metabolic level. The relative mid-parent heterosis in the F(1) hybrids was <20% for most metabolic traits. The first mapping population consisting of 369 recombinant inbred lines (RILs) and their test cross progeny with both parents allowed us to determine the position and effect of 147 quantitative trait loci (QTL) for metabolite absolute mid-parent heterosis (aMPH). Furthermore, we identified 153 and 83 QTL for augmented additive (Z(1)) and dominance effects (Z(2)), respectively. We identified putative candidate genes for these QTL using the aracyc database (http://www.arabidopsis.org/biocyc), and calculated the average degree of dominance, which was within the dominance and over-dominance range for most metabolites. Analyzing a second population of 41 introgression lines (ILs) and their test crosses with the recurrent parent, we identified 634 significant differences in metabolite levels. Nine per cent of these effects were classified as over-dominant, according to the mode of inheritance. A comparison of both approaches suggested epistasis as a major contributor to metabolite heterosis in Arabidopsis. A linear combination of metabolite levels was shown to significantly correlate with biomass heterosis (r = 0.62).

Ethidium Bromide-induced Mutations from Inflorescence Cultures of Indiangrass

Immature inflorescences of a Sorghastrum nutans (L.) Nash selection were cultured on CCm medium with 5 mg·L−1 2,4-dichlorophenoxyacetic acid and 1 mg·L−1 N6-benzyladenine (BA) for 5 weeks. Callused inflorescence cultures were placed on CCm medium with 1 mg·L−1 BA (CCmB1) and 0 or 250 mg·L−1 ethidium bromide (EtBr) for 24 h. Cultures were transferred to CCmB1 without EtBr for shoot regeneration and then to CCm without plant growth regulators for rooting. Rooted shoots were transferred to soil under greenhouse conditions and then to the field. Fifteen putative M1 mutants with atypical phenotypes were detected among 71 EtBr-treated regenerants. Two self-incompatible putative M1 mutants were progeny-tested by using a wild-type Indiangrass seedling as the pollen parent. M1 selection ISU06-35 was a dwarf mutant whose M2 testcross progeny segregated 1:1 tall:dwarf seedlings. M1 selection ISU06-56 was a red-flowered mutant whose M2 testcross progeny segregated 1:1 green-flowered:red-flowered seedlings. These results are consistent with both M1 mutants being dominant nuclear mutations.

Regional mutagenesis using Dissociation in maize

We describe genetic screens, molecular methods and web resources newly available to utilize Dissociation ( Ds) as an insertional mutagen in maize. Over 1700 Ds elements have been distributed throughout the maize genome to serve as donor elements for local or regional mutagenesis. Two genetic screens are described to identify Ds insertions in genes-of-interest (goi). In scheme I, Ds is used to generate insertion alleles when a recessive reference allele is available. A Ds insertion will enable the cloning of the target gene and can be used to create an allelic series. In scheme II, Ds insertions in a goi are identified using a PCR-based screen to identify the rare insertion alleles among a population of testcross progeny. We detail an inverse PCR protocol to rapidly amplify sequences flanking Ds insertion alleles and describe a high-throughput 96-well plate-based DNA extraction method for the recovery of high-quality genomic DNA from seedling tissues. We also describe several web-based tools for browsing, searching and accessing the genetic materials described. The development of these Ds insertion lines promises to greatly accelerate functional genomics studies in maize.

A Comparative Analysis of Conventional and Marker‐Assisted Selection Methods in Breeding Maize Streak Virus Resistance in Maize

Reliable information regarding comparative advantage of marker‐assisted selection (MAS) over conventional selection (CS) in breeding for maize streak virus (MSV) resistance in maize (Zea mays L.) is scarcely available. A comparative study was, therefore, conducted to determine the efficiency of both methods in breeding for MSV resistance in Uganda. Backcross and selfed‐progenies were derived from inbred lines CML202 (resistant), CML321, and CML384 (susceptible) using MAS and CS. The experimental lines and their testcross progenies were evaluated for MSV resistance and yield across three locations. Although both breeding approaches were effective in generating MSV‐resistant lines, disease incidence was higher in populations under CS (79%) than MAS (62%). A similar trend was observed for area under disease progress curve. However, an equal number of lines generated by MAS and CS displayed high yield potential and MVS resistance in testcrosses. Because all required DNA analysis was performed in an existing laboratory and on a well‐characterized quantitative trait locus, costs of capital, equipment maintenance, and marker development were excluded in costing the MAS procedure. Considering total running costs, MAS was cheaper than CS by 26%, which was realized by using fewer plants. Therefore, when laboratory facilities are already established MAS would be recommended in breeding for MSV resistance.

Expression of sunflower cytoplasmic male sterility-associated open reading frame, orfH522 induces male sterility in transgenic tobacco plants

Sterility in the universally exploited PET1-CMS system of sunflower is associated with the expression of orfH522, a novel mitochondrial gene. Definitive evidence that ORFH522 is directly responsible for male sterility is lacking. To test the hypothesis that ORFH522 is sufficient to induce male sterility, a set of chimeric constructs were developed. The cDNA of orfH522 was cloned in-frame with yeast coxIV pre-sequence, and was expressed under tapetum-specific promoter TA29 (construct designated as TCON). For developing control vectors, orfH522 was cloned without the transit peptide under TA29 promoter (TON) or orfH522 was cloned with or without transit peptide under the constitutive CaMV35S promoter (SCOP and SOP). Among several independent transformants obtained with each of the gene cassettes, one third of the transgenics (6/17) with TCON were completely male sterile while more than 10 independent transformants obtained with each of the control vectors were fertile. The male sterile plants were morphologically similar to fertile plants, but had anthers that remained below the stigmatic surface at anthesis. RT-PCR analysis of the sterile plants confirmed the anther-specific expression of orfH522 and bright-field microscopy demonstrated ablation of the tapetal cell layer. Premature DNA fragmentation and programmed cell death was observed at meiosis stage in the anthers of sterile plants. Stable transmission of induced male sterility trait was confirmed in test cross progeny. This constitutes the first report at demonstrating the induction of male sterility by introducing orfH522 gene that could be useful for genetic engineering of male sterility.