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.     

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     

Marker-assisted development and evaluation of near-isogenic lines for scab resistance QTLs of wheat

Fusarium head blight or scab resistance in wheat is a complex quantitative trait affected greatly by environments. Therefore, the quantitative trait loci (QTL) for scab resistance found in mapping projects require validation to be effectively utilized in breeding programs. In this study, by employing both forward and background selections with the help of molecular markers, near-isogenic lines (NILs) for scab resistance QTLs Qfh.nau-2B, Qfhs.nau-3B, Qfhi.nau-4B and Qfhi.nau-5A, three of which originating in scab resistance germplasm Wangshuibai, were developed with the elite line Miangyang 99-323 as the recurrent parent. During the process of backcross, selection was based solely on marker genotypes of the target regions, and on recipient genome recovery rate in BC₂F₁ and BC₃F₁. All the identified BC₃F₁ plants with the target QTL regions have more than 94% recipient genome composition (RGC), and out of four to five of them a plant with over 97% RGC were obtained in each backcross combination. Compared with Mianyang 99-323, the Qfhs.nau-3B NIL showed much better resistance to disease spread within spikes, the Qfhi.nau-4B and Qfhi.nau-5A NILs showed much better resistance to initial infection, and the Qfh.nau-2B NIL showed improvement in both types of resistance. These results were consistent with findings in the previous QTL mapping studies. Morphologically and agronomically these NILs were similar to Mianyang 99-323 except that Qfhi.nau-4B NIL was taller and had a longer spike, and Qfhi.nau-5A NIL had narrower leaves. These results demonstrated the feasibility of marker-assisted utilization of scab resistance QTLs.     

Canopy photosynthesis and its relationship to plant productivity in near-isogenic cotton lines differing in leaf morphology

A 2-year study was conducted to determine the relationships between plant canopy photosynthesis, canopy light interception, and plant productivity of cotton (Gossypium hirsutum L.) exhibiting differing leaf morphologies. The near-isogenic lines were from a single background (MD 65-11) and represented the leaf shapes Normal (small leaf lobing), Sub-Okra (intermediate leaf lobing), Okra (large leaf lobing), and Super Okra (severe leaf lobing). The F₁ of a cross Normal × Okra (intermediate leaf lobing) and the F₂ (segregating 1:2:1 for Normal Sub-Okra, and Okra, respectively) were also grown. Reduced plant canopies were produced by Okra and Super Okra lines, which translated into increased light penetration to the ground, and hence, in reduced canopy photosynthesis. Integrated canopy photosynthesis (ICAP) was significantly associated with light interception by the plant canopy. Part of the remaining variability in ICAP was associated with confounding factors associated with plant maturity and other unmeasured genotypic factors. Intermediate (F₁ and Sub-Okra) and normal leaf types displayed the largest ICAP values in both years. Lint production was positively related to ICAP (R² = 0.53). The combination of high ICAP values and competitive lint yields indicate that intermediate lobed leaf morphologies offer promise as productive sources of physiological variation for cotton germplasm development.     

Analysis of spike productivity traits in near-isogenic lines of the common wheat cultivar Saratovskaya 29 carrying alien marker genes

Six near-isogenic lines of the wheat cultivar Saratovskaya 29 carrying five marker genes from different species (Triticum compactum L., T. polonicum L., T. petropavlovskyi Udacz. et Migusch., Aegilops elongatum Host. and Secale cereale L.) were studied. It was shown that the introduced marker genes of taxonomic significance, C and P, have strong pleiotropic effects on quantitative traits of the spike productivity.     

Integration of statistical and physiological analyses of adaptation of near-isogenic barley lines

Seven near-isogenic barley lines, differing for three independent mutant genes, were grown in 15 environments in Spain. Genotype x environment interaction (G x E) for grain yield was examined with the Additive Main Effects and Multiplicative interaction (AMMI) model. The results of this statistical analysis of multilocation yield-data were compared with a morpho-physiological characterization of the lines at two sites (Molina-Cano et al. 1990). The first two principal component axes from the AMMI analysis were strongly associated with the morpho-physiological characters. The independent but parallel discrimination among genotypes reflects genetic differences and highlights the power of the AMMI analysis as a tool to investigate G x E. Characters which appear to be positively associated with yield in the germplasm under study could be identified for some environments.     

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.     

Identification and verification of QTLs for agronomic traits using wild barley introgression lines

A set of 39 wild barley introgression lines (hereafter abbreviated with S42ILs) was subjected to a QTL study to verify genetic effects for agronomic traits, previously detected in the BC₂DH population S42 (von Korff et al. 2006 in Theor Appl Genet 112:1221–1231) and, in addition, to identify new QTLs and favorable wild barley alleles. Each line within the S42IL set contains a single marker-defined chromosomal introgression from wild barley (Hordeum vulgare ssp. spontaneum), whereas the remaining part of the genome is exclusively derived from elite spring barley (H. vulgare ssp. vulgare). Agronomic field data of the S42ILs were collected for seven traits from three different environments during the 2007 growing season. For detection of putative QTLs, a two-factorial mixed model ANOVA and, subsequently, a Dunnett test with the recurrent parent as a control were conducted. The presence of a QTL effect on a wild barley introgression was accepted, if the trait value of a particular S42IL was significantly (P < 0.05) different from the control, either across all environments and/or in a particular environment. A total of 47 QTLs were localized in the S42IL set, among which 39 QTLs were significant across all tested environments. For 19 QTLs (40.4%), the wild barley introgression was associated with a favorable effect on trait performance. Von Korff et al. (2006 in Theor Appl Genet 112:1221–1231) mapped altogether 44 QTLs for six agronomic traits to genomic regions, which are represented by wild barley introgressions of the S42IL set. Here, 18 QTLs (40.9%) revealed a favorable wild barley effect on the trait performance. By means of the S42ILs, 20 out of the 44 QTLs (45.5%) and ten out of the 18 favorable effects (55.6%) were verified. Most QTL effects were confirmed for the traits days until heading and plant height. For the six corresponding traits, a total of 17 new QTLs were identified, where at six QTLs (35.3%) the exotic introgression caused an improved trait performance. In addition, eight QTLs for the newly studied trait grains per ear were detected. Here, no QTL from wild barley exhibited a favorable effect. The introgression line S42IL-107, which carries an introgression on chromosome 2H, 17–42 cM is an example for S42ILs carrying several QTL effects simultaneously. This line exhibited improved performance across all tested environments for the traits days until heading, plant height and thousand grain weight. The line can be directly used to transfer valuable Hsp alleles into modern elite cultivars, and, thus, for breeding of improved varieties.     

Multi-environment QTL analyses for drought-related traits in a recombinant inbred population of chickpea (Cicer arientinum L.)

A recombinant inbred line (RIL) population, comprising 181 lines derived from ILC588 × ILC3279, was evaluated in 10 environments across three locations with different moisture gradients. A drought resistance score (DRS) and three phenology traits—plant height (PLHT), days to flowering (DFLR), and days to maturity (MAT)—were recorded along with seven yield-related traits—grain yield (GY), biological yield (BY), harvest index (HI), the number of pods/3 plants (Pod), percentage of empty pods (%Epod), 100 seed weight (100 sw), and seed number/3 plants (SN). Two RILs (152, 162) showed the best GYs and DRSs under stressed and non-stressed environments. The quantitative trait loci (QTLs) analyses detected 93 significant QTLs (LOD ≥ 2.0) across the genome × environment interactions. The highest phenotypic variation (>24 %) was explained by the QTL_DFLR in Terbol-11. Four common possible pleiotropic QTLs on LG3 and LG4 were identified as associated with DFLR, DRS, GY, MAT, HI, SN, and Pod. No significant epistatic interactions were found between these QTLs and the other markers. However, the QTL for DRS was detected as a conserved QTL in three late planting environments. The markers H6C-07 (on LG3) and H5G01 (on LG4) were associated with QTLs for many traits in all environments studied except two. The allele ‘A’ of marker H6C07 (from the tolerant parent ILC588) explained 80 % of the yield increase under late planting and 29.8 % of that under dry environments. Concentrating on LG3 and LG4 in molecular breeding programs for drought could speed up improvement for these traits.     

Use of near-isogenic lines derived by backcrossing or selfing to map qualitative traits

Near-isogenic lines (NILs) are a valuable resource for detecting linkages between qualitative trait loci and molecular markers. Molecular marker studies are expensive and methods that require genotyping fewer individuals, such as the NIL-analysis method, are desirable. We present a theory for using sets of NILs to detect linkages between molecular markers and introgressed loci. The probability that a marker a specific distance from the introgressed gene will have a donor parent allele in a near-isogenic line is a function of the distance between the marker and the gene, and the number of back-crosses and/or selfs used in deriving the NIL. The binomial probability formula is used to calculate the probability of having a donor parent allele at a given marker when sets of NILs are used. The formulae given allow calculation of the probability that a marker is linked to the introgressed gene, as well as the probability that a gene will be successfully detected when using given numbers of NILs, backcrosses, and molecular markers.     

Development of chickpea near-isogenic lines for fusarium wilt

Four pairs of near-isogenic lines (NILs) of chickpea with resistance/susceptibility to Fusarium oxysporum f. sp. ciceris (Foc) have been developed in this study. These lines were produced by searching in advanced recombinant inbred lines (RILs) that are segregating for Foc race 5 based on a phenotypic screening. The sequence tagged microsatellite (STMS) marker TA59, closely linked to wilt resistance genes on linkage group 2 (LG2) of the chickpea map, was used to assist the selection of resistant or susceptible genotypes. The NILs were also characterized for disease reaction to Foc races 1A, 2, 3 and 4. Resistance, susceptibility and slow wilting reactions were found in these NILs. Our results suggest that more than one gene controls the resistance to race 5. Combination of the major gene foc-5 linked to TA59 with other gene/s appears to be required to complete resistance, and the absence of these unknown genes leads to slow wilting reactions. The independent differential responses to races 2 and 3 observed in three NILs could be explained as recombination events. This result suggests that foc-2 and foc-3 are delimiting points at opposite ends of a genomic region that includes the remaining foc genes and the TA59 marker. This set of NILs has great potential for studying the genetics and mechanisms of wilt resistance. In addition, the NIL RIP8-94-11 can be used as differential line for Foc race 3; it showed a clear resistance reaction to race 3 and susceptibility to the other Foc races.     

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.     

Unraveling epistasis with triple testcross progenies of near-isogenic lines

Libraries of near-isogenic lines (NILs) are a powerful plant genetic resource to map quantitative trait loci (QTL). Nevertheless, QTL mapping with NILs is mostly restricted to genetic main effects. Here we propose a two-step procedure to map additive-by-additive digenic epistasis with NILs. In the first step, a generation means analysis of parents, their F₁ hybrid, and one-segment NILs and their triple testcross (TTC) progenies is used to identify in a one-dimensional scan loci exhibiting QTL-by-background interactions. In a second step, one-segment NILs with significant additive-by-additive background interactions are used to produce particular two-segment NILs to test for digenic epistatic interactions between these segments. We evaluated our approach by analyzing a random subset of a genomewide Arabidopsis thaliana NIL library for growth-related traits. The results of our experimental study illustrated the potential of the presented two-step procedure to map additive-by-additive digenic epistasis with NILs. Furthermore, our findings suggested that additive main effects as well as additive-by-additive digenic epistasis strongly influence the genetic architecture underlying growth-related traits of A. thaliana.     

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.     

Molecular mapping of QTLs for fiber qualities in three diverse lines in Upland cotton using SSR markers

The improvement of cotton fiber quality is extremely important because of changes in spinning technology. The identification of the stable QTLs affecting fiber traits across different generations will be greatly helpful to be used effectively in molecular marker-assisted selection to improve fiber quality of cotton cultivars in the future. Using three elite fiber lines of Upland cotton (Gossypium hirsutum L.) as parents, three linkage maps were constructed to tag QTLs for fiber qualities using SSR markers. There were 39 QTLs, 17 significant QTLs, LOD 3.0 and 22 suggestive QTLs, 3.0 > LOD 2.0, detected by composite interval mapping for fiber traits, in which 11 QTLs were for fiber length, 10 for fiber strength, 9 for micronaire and 9 for fiber elongation. Out of 17 significant QTLs, 5 QTLs with high logarithm of odds (LOD) score value and stable effect could be found in both F₂ and F_2:3 segregating populations, showing a great potential for molecular-assisted selection in improving fiber quality. At least three common QTLs could be identified in two populations. These common QTLs detected in different populations suggested that there existed elite fiber genes and possibly of the same origin. In addition, we found three pairs of putative homoeologous QTLs, qFL-7-1c and qFL-16-1c, qFS-D03-1a, qFS-A02-1b and qFS-A02-1c, and qFE–D03-1a and qFE-A02-1c. Our results provided a better understanding of the genetic factors of fiber traits in AD tetraploid cottons.     

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.     

Development and characterization of near-isogenic lines of sorghum segregating for glycinebetaine accumulation

Glycinebetaine (GB) is a compatible solute synthesized by species in a number of plant families, including some grasses such as sorghum ( Sorghum bicolor L. Moench). A recombinant-inbred (RI) population was developed from a cross between GB-deficient (IS2319) and GB-accumulating (P932296) sorghum genotypes. The proportions of GB-accumulating lines in both the F₅ and F_7:8 generations of this RI population were higher than expected, possibly due to some beneficial effect of GB accumulation on plant growth, survival or seed set. GB levels varied widely in lines of the RI population that were grown under controlled conditions, suggesting genetic control not only for the presence or absence of GB, but also for the level of GB. This hypothesis was tested by analysing individual plants from lines identified as accumulating low, medium or high concentrations of GB based on the F₅ and F_7:8 screens. The level of GB was conserved within lines, supporting the hypothesis of genetic control of relative GB levels within accumulating lines. Two pairs of near-isogenic lines (NILs) with contrasting GB levels within pairs were developed from the RI population. The stable inheritance of the GB phenotype and isogenicity of these NILs were confirmed with progeny tests and molecular marker analysis, respectively. Labelling studies demonstrated that the deficiency in GB accumulation was at the choline oxidation step.     

Individual and multi-environment combined analyses identify QTLs for morphogenetic and reproductive development traits in white clover (Trifolium repens L.)

White clover (Trifolium repens L.) is a key component legume of temperate pasture agriculture and an important target for molecular marker-assisted plant breeding. A genetic map of white clover has been used to assess genetic control of agronomically important traits that vary in the F₂(I.4R×I.5J) mapping family. Phenotypic analysis was performed for a range of vegetative morphogenesis traits (such as leaf area, internode length, plant height and plant spread) and reproductive morphogenesis and development traits (such as flowering date, floral intensity and seed yield), with both spatial and temporal replication. A multi-environment combined analysis (combined analysis) has been performed for traits assessed across multiple experimental datasets in order to identify consistent genetic effects. Quantitative trait locus (QTLs) were detected for the majority of traits, and the locations and magnitudes of QTL effects were compared between individual and combined analyses. This molecular genetic dissection of agronomic traits in white clover provides the basis for equivalent studies in more complex populations, design of marker-assisted selection strategies and comparative genetics with model legume species. Selection for QTLs derived from the combined analysis will permit robust improvement of phenotypic traits over different environments.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.