Development of wheat near-isogenic lines for powdery mildew resistance

Using three Chinese wheat cultivars, Bainong 3217, Beijing 837 and Laizhou 953, as recurrent parents, 33 near-isogenic lines (NILs) carrying 22 powdery mildew resistance genes (Pm1c, Pm2, Pm4b, Pm12, Pm13, Pm16, Pm20, Pm21, Pm23, and 13 undocumented genes) were developed. All NILs had no significant difference to their recurrent parents in the investigated traits of agronomic importance. The results of AFLP analysis indicated Jaccards genetic similarity of the NILs with their recurrent parents varied from 0.96 to 0.98, and confirmed that the NILs had high genetic similarity with their recurrent parents. The resistance to powdery mildew was stably expressed by the relevant NILs. Eleven of the NILs were tested using molecular markers linked to the resistance genes Pm1c, Pm4b, Pm13, Pm21, PmP, PmE, PmPS5A, PmPS5B, PmY39, PmY150, and PmH, and they were all found to carry the targeted genes. The potential application of these NILs in gene discovery is discussed.     

Field evaluation of cotton near-isogenic lines introgressed with QTLs for productivity and drought related traits

Quantitative trait loci (QTLs) for yield and drought related physiological traits, osmotic potential (OP), carbon isotope ratio (δ¹³C, an indicator of water use efficiency), and leaf chlorophyll content (Chl), were exchanged via marker-assisted selection (MAS) between elite cultivars of the two cotton species Gossypium barbadense cv. F-177 and G. hirsutum cv. Siv’on. The resulting near isogenic lines (NILs) were examined in two field trials, each with two irrigation regimes, in order to (1) evaluate the potential to improve cotton drought resistance by MAS and (2) test the role of physiological traits in plant productivity. NILs introgressed with QTLs for high yield rarely exhibited an advantage in yield relative to the recipient parent, whereas a considerable number of NILs exhibited the expected phenotype in terms of lower OP (5 out of 9), higher δ¹³C (4 out of 6) or high Chl (2 out of 3). Several NILs exhibited considerable modifications in non-targeted traits including leaf morphology, stomatal conductance and specific leaf weight (SLW). In G. barbadense genotypes, yield was correlated negatively with δ¹³C and OP and positively with stomatal conductance, SLW and Chl, whereas in G. hirsutum yield was negatively correlated with δ¹³C, SLW and Chl. This dissimilarity suggests that each of the respective species has evolved different mechanisms underlying plant productivity. We conclude that the improvement of drought related traits in cotton NILs may lead to improved drought resistance via MAS, but that conventional breeding may be necessary to combine the introduced QTL(s) with high yield potential.     

Marker-assisted characterization of Asian wheat lines for resistance to Fusarium head blight

The major quantitative trait locus (QTL) on 3BS from Sumai 3 and its derivatives has been used as a major source of resistance to Fusarium head blight (FHB) worldwide, but resistance genes from other sources are necessary to avoid complete dependence on a single source of resistance. Fifty-nine Asian wheat landraces and cultivars differing in the levels of FHB resistance were evaluated for type II FHB resistance and for genetic diversity on the basis of amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSRs). Genetic relationships among these wheat accessions estimated by cluster analysis of molecular marker data were consistent with their geographic distribution and pedigrees. Chinese resistant landraces had broader genetic diversity than that of accessions from southwestern Japan. The haplotype pattern of the SSR markers that linked to FHB resistance quantitative trait loci (QTLs) on chromosomes 3BS, 5AS and 6BS of Sumai 3 suggested that only a few lines derived from Sumai 3 may carry all the putative QTLs from Sumai 3. About half of the accessions might have one or two FHB resistance QTLs from Sumai 3. Some accessions with a high level of resistance, may carry different FHB resistance loci or alleles from those in Sumai 3, and are worth further investigation. SSR data also clearly suggested that FHB resistance QTLs on 3BS, 5AS, and 6BS of Sumai 3 were derived from Chinese landrace Taiwan Xiaomai.     

Aphid (Hemiptera: Aphididae) resistance in wheat near-isogenic lines

Plant and aphid biomass, photosynthetic pigment (chlorophylls a and b and carotenoids) concentrations, and chlorophyll a/b and chlorophyll/carotenoid ratios were quantified in aphid-infested 'Tugela' near-isogenic lines (Tugela, Tugela-Dn1, Tugela-Dn2, and Tugela-Dn5). The objectives were to quantify changes of photosynthetic pigments (chlorophylls a and b, and carotenoids) caused by aphid feeding and assess resistance of wheat isolines through aphid and plant biomass analysis. Biomass of bird cherry-oat aphid, Rhopalosiphum padi (L.) (Hemiptera: Aphididae)-infested plants was lower than Russian wheat aphid, Diuraphis noxia (Mordvilko) (Hemiptera: Aphididae),- infested plants. When infested by D. noxia, all lines showed increased biomass over time, except Tugela where biomass decreased on day 12. No difference in plant biomass was detected among R. padi-infested and uninfested wheat lines. Biomass of D. noxia from Tugela (D. noxia-susceptible) was significantly higher than from plants with Diuraphis noxia-resistant Dn genes. Diuraphis noxia biomass from Tugela-Dn1 and Dn2 lines was not different from each other, but they were lower than from Tugela-Dn5. In contrast, there was no difference in R. padi biomass among wheat lines. Concentrations of chlorophylls a and b and carotenoids were significantly lower in D. noxia-infested plants compared with R. padi-infested and uninfested plants. When infested by D. noxia, chlorophyll a and b concentrations were not different among wheat lines on day 3, but they were lower in Tugela and Tugela-Dn1 than in Tugela-Dn2 and -Dn5 plants on days 6 and 12. However, no difference was detected in chlorophyll a/b or chlorophyll/carotenoid ratio among Tugela lines. The study demonstrated that Dn genes in the Tugela isolines conferred resistance to D. noxia but not to R. padi. Tugela-Dn1 was antibiotic, Tugela-Dn2 was tolerant and antibiotic, and Tugela-Dn5 was moderately antibiotic.     

Mapping QTLs that control the performance of rice tissue culture and evaluation of derived near-isogenic lines

Quantitative trait loci (QTLs) that control the performance of tissue culture in rice were detected by using 116 RFLP markers and 183 BC₁F₃ lines derived from two varieties, Koshihikari and Kasalath. With time, the seed callus of Koshihikari tends to turn brown and stop growing, while that of Kasalath remains yellowish-white and proliferates continuously. The performance of tissue culture in the induction of calli from seed, the subculture of induced calli, and shoot regeneration were evaluated by five indices: induced-callus weight, induced-callus color, subcultured-callus volume, subcultured-callus color, and regeneration rate. Through callus induction and subculture, eight putative QTLs (P < 0.001) were located on chromosomes 1, 4, and 9. Among these QTLs, five Kasalath alleles and three Koshihikari alleles improved tissue culture performance. No QTL for regeneration was found. Among all the QTLs, qSv1 explained the largest phenotypic variance, 33%, in subcultured-callus volume. In induced-callus color, two detected QTLs accounted for 36.4% of the total phenotypic variance; this was the highest score among the five indices used to evaluate the performance of tissue culture. Three near-isogenic lines for QTLs, located in two regions on chromosome 1, were developed to evaluate their tissue culture performance. The Kasalath alleles in qSv1 and qSc1-1 improved callus color through callus induction and subculture, and increased the subcultured-callus volume and the fresh weight of regenerated calli, including shoots, roots, and differentiated structures. In qSc1-2, the Kasalath allele improved callus color through induction and subculture. These results verified the presence of QTLs for the volume and color of subcultured callus on chromosome 1, qSv1, qSc1-1, and qSc1-2.     

Marker-assisted recurrent selection for cumulating additive and interactive QTLs in recombinant inbred lines

A computer program has been designed to manage marker information in recombinant inbred-line populations. The objective is to select pairs of inbred lines (either recombinant-inbred or doubled-haploid) to be intercrossed, in order to accumulate all or most favourable alleles, either with additive effects or with interactive effects. The population size required to have a 95% chance of obtaining the best line from a given cross is computed, taking into account the number of QTLs and the probability that no recombination event occurs in any of the QTL confidence intervals. It is shown that the accuracy of QTL location greatly affects selection efficiency and that a recurrent selection scheme is highly preferable for pyramiding many QTLs. An application to the bread-making quality improvement of wheat is presented. Received: 25 September 1998 / Accepted: 29 July 1999     

Marker-assisted development and characterization of near-isogenic lines carrying the Rx4 gene for hypersensitive resistance to Xanthomonas euvesicatoria pv. perforans race T3 in tomato

Molecular Breeding - Stigma exsertion is a plant trait wherein stigmas emerge from flower buds before anthesis. It is a key determinant of the ornamental kale mating system, greatly increases the...     

Rooting patterns in near-isogenic lines of spring wheat for dwarfism

The effects of Rht alleles on root growth and distribution in isogenic lines of spring wheat (Triticum aestivum L.) are described under different environmental conditions. Above-ground biomass, root length, root dry-weight and their distribution along the soil profile were measured by destructive sampling for growth of aerial biomass and extraction of soil cores containing roots. Field experiments were conducted under non-limiting water and nutritional conditions during two consecutive years, using an early and a late sowing date each year.Dwarfing genes significantly reduced plant height and above-ground biomass at anthesis. In addition, stem mass ratio also was reduced with increases in the allelic dosage. Conversely, total root length and root dry-weight per unit area at anthesis were increased with decreased plant height, therefore, root mass ratio tended to be negatively correlated with plant height. Differences in distribution of root length and root dry-weight through the soil profile among lines were largely confined to the upper soil layers (i.e. the top 30 cm).Differences in root dry-weight were more important than in root length, so that the dwarf line had the highest root mass per unit root length. Furthermore, a significant positive correlation between the root mass ratio and stem mass per unit stem length was found. It is suggested that increases in root mass per unit root length associated with Rht alleles are evidencing a surplus of photoassimilates during stem elongation which are used for thickening the roots due to the lack of alternative sinks. Agronomic implications of this effect are discussed.     

Metabolite and mineral analyses of cotton near-isogenic lines introgressed with QTLs for productivity and drought-related traits

Quantitative trait loci (QTLs) for yield and drought-related traits were exchanged via marker-assisted selection between elite cultivars of two cotton species, Gossypium barbadense (GB) cv. F-177 and Gossypium hirsutum (GH) cv. Siv'on. Three of the resultant near-isogenic lines (NILs), each introgressed with a different QTL region, expressed an advantage in osmotic adjustment (OA) and other drought-related traits relative to their recipient parents. These NILs and the parental genotypes were field-grown under well-watered and water-limited conditions, and characterized for their metabolic and mineral compositions. Comparisons were then made between (1) GB and GH genotypes, (2) the contrasting water regimes and (3) each NIL and its recipient parent. Hierarchical clustering analysis clearly distinguished between GB and GH genotypes based on either metabolite or mineral composition. Comparisons between well-watered and water-limited conditions in each of the genotypes showed differing trends in the various solutes. The greater concentrations of potassium, magnesium and calcium under water stress, when compared with well-watered conditions, may have enhanced OA or osmoprotection. All NILs exhibited significantly modified solute composition relative to their recipient parents. In particular, increased levels of alanine, aspartic acid, citric acid, malic acid, glycerol, myoinositol, threonic acid, potassium, magnesium and calcium were found under drought conditions in one or more of the NILs relative to their recipient parents. The increased values of these solutes could contribute to the superior capacity of these NILs to cope with drought.     

Evaluation of near-isogenic lines of rice introgressed with QTLs for root depth through marker-aided selection

Drought is one of the main abiotic constraints in rice. A deep root system contributes efficiently to maintaining the water status of the crop through a stress period. After identifying QTLs affecting root parameters in a doubled-haploid (DH) population of rice derived from the cross IR64/Azucena, we started a marker-assisted backcross program to transfer the Azucena allele at four QTLs for deeper roots (on chromosomes 1, 2, 7 and 9) from selected DH lines into IR64. We selected the backcross progenies strictly on the basis of their genotypes at the marker loci in the target regions up to the BC₃F₂. We assessed the proportion of alleles remaining from Azucena in the non-target areas of the BC₃F₂ plants, which was in the range expected for the backcross stage reached. Twenty nine selected BC₃F₃ near-isogenic lines (NILs) were developed and compared to IR64 for the target root traits and three non-target traits in replicated experiments. Of the three tested NILs carrying target 1, one had significantly improved root traits over IR64. Three of the seven NILs carrying target 7 alone, as well as three of the eigth NILs carrying both targets 1 and 7, showed significantly improved root mass at depth. Four of the six NILs carrying target 9 had significantly improved maximum root length. Five NILs carrying target 2 were phenotyped, but none had a root phenotype significantly different from that of IR64. A re-analysis of the initial data with the composite interval mapping technique revealed two linked QTLs with opposite effects in this area. Some NILs were taller than IR64 and all had a decreased tiller number because of a likely co-introgression of linked QTLs. The usefulness of NILs, the efficiency of marker-aided selection for QTLs and the relationship between root traits are discussed. The NILs with an improved root system will permit testing the importance of root depth for water-limited environments. Received: 17 July 2000 / Accepted: 20 October 2000     

Application of STS markers for leaf rust resistance genes in near-isogenic lines of spring wheat cv. Thatcher

Sequence tagged site (STS) markers for eight resistance genes against Puccinia recondita f. sp. tritici were used to screen a set of near-isogenic lines of wheat cv. Thatcher containing in total 40 different Lr genes and their alleles. Polymerase chain reaction (PCR) analysis was carried out by using STS, SCAR and CAPS primers specific for the leaf rust resistance genes Lr1, Lr9, Lr10, Lr19, Lr24, Lr28, Lr37 and Lr47. The STS, CAPS and SCAR markers linked to resistance genes Lr9, Lr10, Lr19, Lr24, Lr37 and Lr47 were found to be reliable in diverse genetic backgrounds. The amplification product of the Lr1 gene marker was detected in the susceptible cv. Thatcher and in all of the near-isogenic lines examined except Lr2a, Lr2b, Lr2c and Lr19. The sequence analysis of PCR products amplified in lines Lr1, Lr10, Lr28 and in cv. Thatcher indicated that the near-isogenic lines and cv. Thatcher contained in the targeted chromosome region an allele that differed from the original alleles corresponding to Lr1/6*Thatcher (TLR621) and susceptible Thatcher (TH621). The amplification product specific to the STS marker of the Lr1 gene was amplified in almost all Thatcher near-isogenic lines and in cv. Thatcher because their alleles possessed primer sequences identical to the original allele TLR621. The marker for the Lr28 resistance gene was identified in line Lr28, carrying gene Lr28, and in 21 other near-isogenic lines. The sequencing of PCR products specific to Lr28 and generated in lines Lr1, Lr10 and Lr28 indicated that the lines Lr1, Lr10 and Lr28 are heterozygous in this region.     

Cold hardiness of wheat near-isogenic lines differing in vernalization alleles

Four major genes in wheat (Triticum aestivum L.), with the dominant alleles designated Vrn-A1, Vrn-B1, Vrn-D1, and Vrn4, are known to have large effects on the vernalization response, but the effects on cold hardiness are ambiguous. Near-isogenic experimental lines (NILs) in a Triple Dirk (TD) genetic background with different vernalization alleles were evaluated for cold hardiness. Although TD is homozygous dominant for Vrn-A1 (formerly Vrn1) and Vrn-B1 (formerly Vrn2), four of the lines are each homozygous dominant for a different vernalization gene, and one line is homozygous recessive for all four vernalization genes. Following establishment, the plants were initially acclimated for 6 weeks in a growth chamber and then stressed in a low temperature freezer from which they were removed over a range of temperatures as the chamber temperature was lowered 1.3°C h^–1. Temperatures resulting in no regrowth from 50% of the plants (LT₅₀) were determined by estimating the inflection point of the sigmoidal response curve by nonlinear regression. The LT₅₀ values were –6.7°C for cv. TD, –6.6°C for the Vrn-A1 and Vrn4 lines, –8.1°C for the Vrn-D1 (formerly Vrn3) line, –9.4°C for the Vrn-B1 line, and –11.7°C for the homozygous recessive winter line. The LT₅₀ of the true winter line was significantly lower than those of all the other lines. Significant differences were also observed between some, but not all, of the lines possessing dominant vernalization alleles. The presence of dominant vernalization alleles at one of the four loci studied significantly reduced cold hardiness following acclimation.     

Marker-assisted introgression of five QTLs controlling fruit quality traits into three tomato lines revealed interactions between QTLs and genetic backgrounds

The evaluation of organoleptic quality of tomato fruit requires physical, chemical and sensory analyses, which are expensive and difficult to assess. Therefore, their practical use in phenotypic selection is difficult. In a previous study, the genetic control of several traits related to organoleptic quality of fresh-market tomato fruit was investigated. Five chromosome regions strongly involved in organoleptic quality attributes were then chosen to be introgressed into three different recipient lines through marker-assisted selection. A marker-assisted backcross (MABC) strategy was performed, as all the favorable alleles for quality traits were provided by the same parental tomato line, whose fruit weight (FW) and firmness were much lower than those of the lines commonly used to develop fresh market varieties. Three improved lines were obtained after three backcrossing and two selfing generations. The implementation of the MABC scheme is described. The three improved lines were crossed together and with the recipient lines in a half-diallel mating scheme, and the simultaneous effect of the five quantitative trait locus (QTL) regions was compared in different genetic backgrounds. Significant effects of the introgressed regions and of the genetic backgrounds were shown. Additive effects were detected for soluble solid and reducing sugar content in two genetic backgrounds. A partially dominant effect on titratable acidity was detected in only one genetic background. In contrast, additive to dominant unfavorable effects of the donor alleles were detected for FW and locule number in the three genetic backgrounds. Recessive QTL effects on firmness were only detected in the two firmest genetic backgrounds. Comparison of the hybrids in the half-diallel gave complementary information on the effects of: (1) the alleles at the selected regions, (2) the genetic backgrounds and (3) their interaction. Breeding efficiency strongly varied according to the recipient parent, and significant interactions between QTLs and genetic backgrounds were shown for all of the traits studied.     

The evaluation of FHB resistance QTLs introgressed into elite Canadian spring wheat germplasm

FHB resistance QTL alleles from Nyuubai, Sumai-3, and Wuhan-1 were evaluated for their effect on Fusarium head blight (FHB) index, Fusarium damaged kernels (FDK), deoxynivalenol (DON) accumulation, plant height, anthesis date, and numerous grain quality traits in three elite Canadian spring wheat backgrounds. The three FHB resistance parameters were negatively correlated with plant height in the three populations. The Wuhan-1 4B resistance allele was the most effective resistance allele but was associated with a 9.3 cm increase in plant height. The Wuhan-1 2D, Nyuubai 3BSc, Sumai-3 3BSc, Nyuubai 5AS, and Sumai-3 5AS alleles were also effective FHB resistance alleles in these populations. The Nyuubai and Sumai-3 3BS alleles were the least effective of the FHB resistance alleles in the FHB nursery tests. The Sumai-3 5AS resistance allele was significantly associated with reduced grain protein content, while the same trend was observed for the Nyuubai 5AS resistance allele but was not significant. FHB resistance tended to increase with more FHB resistance alleles introgressed into the elite genetic background, which suggested that marker-assisted selection (MAS) will prove useful for improving FHB resistance in Canadian germplasm.     

Characterization of disease resistance gene-like sequences in near-isogenic lines of tomato

 We examined near-isogenic lines (NILs) carrying either of the tomato mosaic virus (ToMV) resistance genes Tm-1 and Tm-2 for sequences homologous to the isolated disease-resistance genes. DNA fragments were amplified from the genomic DNA of the NILs by the polymerase chain reaction (PCR) using primers designed on the basis of sequences of certain domains conserved among some disease-resistance genes. Of ten PCR products cloned, five were identified as having homology to either of the two classes of disease-resistance genes. The first class encoded proteins containing leucine-rich repeats (LRRs) and a nucleotide-binding site (NBS), such as the RPS2 gene in Arabidopsis and the N gene in tobacco. The second class encoded proteins containing a C-terminal membrane anchor but no NBS, such as the Cf 2 and Cf 9 genes in tomato. In Southern hybridization of the genomic DNAs of the NILs carrying either Tm-1 or Tm-2 and their parental NIL carrying neither of these resistance genes, multiple bands could be detected with most of the clones used as probes. This suggests that the genomes of the NILs contain multiple copies of sequences homologous to some of the known disease-resistance genes. No evidence was obtained to show that the Tm-1 and/or Tm-2 loci encode either class of protein, since no polymorphic band patterns between the NILs were detected by Southern hybridization. Received: 15 August 1997 / Accepted: 2 September 1997     

Differential accumulation of cadmium in near-isogenic lines of durum wheat: no role for phytochelatins

Certain cultivars of some crops, including durum wheat (Triticum durum Desf.), have a propensity to accumulate cadmium in the grain. In the 1980s, a Canadian wheat breeding program generated five pairs of near-isogenic lines of durum wheat that vary in cadmium-accumulation. Within each pair, one member accumulates twofold to threefold higher concentrations of cadmium in the shoot and grain. However, the physiological explanation for the high-low phenotype is unknown. We studied correlations between concentrations of cadmium and non-protein thiols, including phytochelatins, in these five pairs of near-isogenic lines to test the hypothesis that differential retention of cadmium-binding complexes in the root would explain the phenotype. The expected high-low pattern of cadmium accumulation was found in three of the pairs. In one pair, cadmium was positively correlated with cysteine and glutathione in the roots and with phytochelatins 2 and 4 in the shoots but in another pair cadmium was strongly negatively correlated with phytochelatins 2 and 4 in the shoots and unrelated to cysteine or glutathione. No correlations between concentrations of cadmium and the non-protein thiols were found in the third pair or in the remaining two pairs. The production of phytochelatins is a well-described response to cadmium but the lack of consistent correlation between cadmium and non-protein thiols in these five near-isogenic lines indicates that complexation with non-protein thiols does not explain differential translocation of cadmium in durum wheat.     

Cloning a DNA marker associated to wheat scab resistance

Wheat head blight caused mainly by Fusarium graminearum, is an important wheat disease, causing yield and quality losses. The breeding of resistant varieties is the key measure to control this disease, but the conventional breeding method is of low efficiency. The marker-assisted selection (MAS) can significantly improve the breeding efficiency. In this study, four RAPD (randomly amplified polymorphic DNA) markers linked to FHB resistance were obtained and one was cloned and sequenced. F7 recombinant inbred lines (RILs) were derived from the F1 of the cross Ning894037 (resistant)/Alondra (susceptible) by the single-seed descent method. Scab resistance of F7 RILs was evaluated in the greenhouse by injecting conidiospores into a central floret. Scab symptoms were evaluated on the 21st day after inoculation. Disease severity was assessed as the percentage of infected spikelets/spike. The F7 RIL population displayed a normal distribution, transgressive segregation and significant variation for FHB severity. DNA from resistant and susceptible parents was analyzed with 520 RAPD primers. Four markers (S1384-640,S1360-600, S1319-350,S1319-820) linked to FHB resistance were obtained. DNA of S1384-640 was recovered, subjected to re-amplification by using S1384 primer and the same protocol as for RAPD analysis and identified the rightness. The PCR product of S1384-640 was ligated into the pUCm-T vector, and cloned into fresh competent cells of Escherichia coli strain DH5alpha RAPD analysis showed that the inserts of the recombinant plasmids were DNA of S1384-640. The sequencing result showed that the cloned fragment was 648 bp.