Extensive genomic characterization of a set of near-isogenic lines for heterotic QTL in maize (Zea mays L.).

BACKGROUND: Despite the crucial role that heterosis has played in crop improvement, its genetic and molecular bases are still elusive. Several types of structured populations were used to discover the genetic architecture underlying complex phenotypes, and several QTL related to heterosis were detected. However, such analyses generally lacked the statistical power required for the detailed characterization of individual QTL. Currently, QTL introgression into near-isogenic materials is considered the most effective strategy to this end, despite such materials inevitably contain a variable, unknown and undesired proportion of non-isogenic genome.An introgression program based on residual heterozygous lines allowed us to develop five pairs of maize (Zea mays L.) near-isogenic lines (NILs) suitable for the fine characterization of three major heterotic QTL previously detected. Here we describe the results of the detailed genomic characterization of these NILs that we undertook to establish their genotypic structure, to verify the presence of the expected genotypes within target QTL regions, and to determine the extent and location of residual non-isogenic genomic regions. RESULTS: The SNP genotyping approach allowed us to determine the parent-of-origin allele for 14,937 polymorphic SNPs and to describe in detail the genotypic structure of all NILs. The correct introgression was confirmed for all target QTL in the respective NIL and several non-isogenic regions were detected genome-wide. Possible linkage drag effects associated to the specific introgressed regions were observed. The extent and position of other non-isogenic regions varied among NIL pairs, probably deriving from random segregating sections still present at the separation of lineages within pairs. CONCLUSIONS: The results of this work strongly suggest that the actual isogenicity and the genotypic architecture of near-isogenic materials should be monitored both during the introgression procedure and on the final materials as a paramount requisite for a successful mendelization of target QTL. The information here gathered on the genotypic structure of NILs will be integrated in future experimental programs aimed at the fine mapping and isolation of major heterotic QTL, a crucial step towards the understanding of the molecular bases of heterosis in maize.

Divergent selection in a maize population for germination at low temperature in controlled environment: study of the direct response, of the trait inheritance and of correlated responses in the field.

Improving cold tolerance in maize (Zea mays L.) is an important breeding objective, allowing early sowings which result in many agronomic advantages. Using as source the F(2) population of B73 × IABO78 single cross, we previously conducted four cycles of divergent recurrent selection for high (H) and low (L) cold tolerance level, evaluated as the difference (DG) between germination at 9.5 °C and at 25 °C in the germinator. Then, we pursued the divergent selection in inbreeding from S(1) to S(4). This research was conducted to study (1) the direct response to selection (by testing ten S(4) L and ten S(4) H lines), (2) the trait inheritance (in a complete diallel scheme involving four L and four H lines), (3) the associated responses for cold tolerance in the field (at early and delayed sowings) and (4) the responses for other traits, by testing the ten L and the ten H lines at usual sowing. Selection was effective, leading to appreciable and symmetric responses for DG. Variation among crosses was mainly due to additive effects and the ability to predict hybrid DG based on parental lines DG was appreciable. Associated responses for cold tolerance traits in the field were noticeable, though the relationship between DG and these traits was not outstanding. High tolerance was also associated with early flowering, short plants, less leaves, low kernel moisture, red and thin cob, and flint kernels. These divergently selected lines can represent valuable materials for undertaking basic studies and breeding works concerning cold tolerance.

Characterization of heterotic quantitative trait loci in maize by evaluation of near-isogenic lines and their crosses at two competition levels.

In a previous study on a maize (Zea mays L.) population of recombinant inbreds derived from B73 × H99, we identified several quantitative trait loci (QTL) for agronomic traits with high dominance-additive ratio. Then, for four of these QTL, we developed families of near-isogenic lines (NILs) homozygous either for the QTL allele from B73 (BB) or from H99 (HH); for two of these QTL, the NILs' families were produced in two different genetic backgrounds. The present study was conducted to: (1) characterize these QTL for agronomic traits and (2) verify whether their effects were influenced by the genetic background, inbreeding level and plant density (PD). The six NILs' families were tested across 3 years and in three experiments at different inbreeding levels as NILs per se and their reciprocal crosses (Experiment 1), NILs crossed to related inbreds B73 and H99 (Experiment 2) and NILs crossed to four unrelated inbreds (Experiment 3). Experiment 2 was conducted at two PDs (4.5 and 9.0 plants m(-2)). Results of Experiments 1 and 2 confirmed previous findings as to QTL effects, with dominance-additive ratio superior to 1 for several traits; as a tendency, dominance effects were more pronounced in Experiment 1. The QTL effects were also confirmed in Experiment 3. The interactions involving QTL effects, families and PD were generally negligible, suggesting a certain stability of the QTL. Results emphasize the importance of dominance effects for these QTL, suggesting that they might deserve further studies, using the NILs' families and their crosses as base materials.

The activity of the plant mitochondrial inner membrane anion channel (PIMAC) of maize populations divergently selected for cold tolerance level is differentially dependent on the growth temperature of seedlings.

The activity of the plant inner membrane mitochondrial anion channel (PIMAC) is involved in metabolite shuttles and mitochondrial volume changes and could have a role in plant temperature tolerance. Our objectives were to investigate (i) the occurrence and (ii) the temperature dependence of anion fluxes through PIMAC in mitochondria isolated from seedlings of three maize populations differing in terms of cold tolerance; and (iii) the relationships between the PIMAC activity kinetics and the level of cold tolerance. Populations were the source population (C0) and two populations divergently selected for high (C4H) and low (C4L) cold tolerance. Such divergently selected populations are expected to share most of their genes, with the main exception of those genes controlling cold tolerance. Arrhenius plots of PIMAC chloride fluxes showed a linear temperature dependence when seedlings were grown at 25 or 14°C, whereas a non-linear temperature dependence was found when seedlings were grown at 5°C, with or without acclimation at 14°C. The activation energy and other thermodynamic parameters of PIMAC activity varied depending on temperature treatments during seedling growth. When seedlings were grown at 14 and 5°C with acclimation, PIMAC activity of the C4H population increased, while that of C4L declined, as compared with the activities of seedlings grown at 25°C. These symmetric responses indicate that PIMAC activity changes are associated with genetically determined differences in the cold tolerance level of the investigated populations. We conclude that anion fluxes by PIMAC depend upon changes on growth temperature and are differentially related to the tolerance level of the tested populations.

Characterization of root-yield-1.06, a major constitutive QTL for root and agronomic traits in maize across water regimes.

A previous study on maize F(2:3) families derived from Lo964xLo1016 highlighted one QTL in bin 1.06 (hereafter named root-yield-1.06) affecting root and agronomic traits of plants grown in well-watered (WW) and water-stressed (WS) conditions. Starting from different F(4) families, two pairs of near isogenic lines (NILs) were developed at root-yield-1.06. The objective of this study was to evaluate root-yield-1.06 effects across different water regimes, genetic backgrounds, and inbreeding levels. The NILs per se and their crosses with Lo1016 and Lo964 were tested in 2008 and 2009 near to Bologna, with the well-watered (WW) and water-stressed (WS) treatments providing, on average, 70 mm and 35 mm of water, respectively. For NILs per se, the interactions QTL x water regime and QTL x family were negligible in most cases; the QTL additive effects across families were significant for several traits, especially root clump weight. For NILs crosses, analogously to NILs per se, the interactions were generally negligible and the additive effects across water regimes and families were significant for most traits, especially grain yield. A meta-analysis carried out considering the QTLs described in this and previous studies inferred one single locus as responsible for the effects on roots and agronomic traits. Our results show that root-yield-1.06 has a major constitutive effect on root traits, plant vigour and productivity across water regimes, genetic backgrounds, and inbreeding levels. These features suggest that root-yield-1.06 is a valuable candidate for cloning the sequence underlying its effects and for marker-assisted selection to improve yield stability in maize.

QTL detection in maize testcross progenies as affected by related and unrelated testers.

The evaluation of recombinant inbred lines (RILs) per se can be biased by inbreeding depression in case of allogamous species. To overcome this drawback, RILs can be evaluated in combination with testers; however, testers can carry dominant alleles at the quantitative trait loci (QTL), thus hampering their detection. This study was conducted on the maize (Zea mays L.) population of 142 RILs derived from the single cross B73 x H99 to evaluate the role of different testers in affecting: (1) QTL detection, (2) the estimates of their effects, and (3) the consistency of such estimates across testers. Testcrosses (TCs) were produced by crossing RILs with inbred testers B73 [TC(B)], H99 [TC(H)], and Mo17 [TC(M)]. TCs were field tested in three environments. TC(B) mean was higher than TC(H) mean for all traits, while TC(M) mean was the highest for plant vigor traits and grain yield. As to the number of detected QTL, tester Mo17 was superior to H99 and B73 for traits with prevailing additive effects. Several overlaps among the QTL were detected in two or all the three TC populations with QTL effects being almost always consistent (same sign). For traits with prevailing dominance-overdominance effects, as grain yield, the poor performing tester H99 was clearly the most effective; fewer overlaps were found and some of them were inconsistent (different sign). Epistatic interactions were of minor importance. In conclusion, the three testers proved to affect QTL detection and estimation of their effects, especially for traits showing high dominance levels.

Recombinant near-isogenic lines: a resource for the mendelization of heterotic QTL in maize.

Although heterosis is widely exploited in agriculture, a clear understanding of its genetic bases is still elusive. This work describes the development of maize recombinant near-isogenic lines (NILs) for the mendelization of six heterotic QTL previously identified based on a maize (Zea mays L.) RIL population. The efficient and inexpensive strategy adopted to generate sets of NILs starting from QTL-specific residual heterozygous lines (RHLs) is described and validated. In particular, we produced nine pairs of recombinant NILs for all six QTL starting from RHLs F(4:5) originally obtained during the production of the RIL population mentioned above. Whenever possible, two different NIL pairs were generated for each QTL. The efficiency of this procedure was tested by analyzing two segregating populations for two of the selected heterotic QTL for plant height, yield per plant and ears per plant. Both additive and dominant effects were observed, consistently with the presence of the QTL within the introgressed regions. Refinement of QTL detection was consistent with previous observations in terms of effects and position of the considered QTL. The genetic material developed in this work represents the starting point for QTL fine mapping aimed at understanding the genetic bases of hybrid vigor in maize.

Classical genetic and quantitative trait loci analyses of heterosis in a maize hybrid between two elite inbred lines.

The exploitation of heterosis is one of the most outstanding advancements in plant breeding, although its genetic basis is not well understood yet. This research was conducted on the materials arising from the maize single cross B73 x H99 to study heterosis by procedures of classical genetic and quantitative trait loci (QTL) analyses. Materials were the basic generations, the derived 142 recombinant inbred lines (RILs), and the three testcross populations obtained by crossing the 142 RILs to each parent and their F(1). For seedling weight (SW), number of kernels per plant (NK), and grain yield (GY), heterosis was >100% and the average degree of dominance was >1. Epistasis was significant for SW and NK but not for GY. Several QTL were identified and in most cases they were in the additive-dominance range for traits with low heterosis and mostly in the dominance-overdominance range for plant height (PH), SW, NK, and GY. Only a few QTL with digenic epistasis were identified. The importance of dominance effects was confirmed by highly significant correlations between heterozygosity level and phenotypic performance, especially for GY. Some chromosome regions presented overlaps of overdominant QTL for SW, PH, NK, and GY, suggesting pleiotropic effects on overall plant vigor.

Root-ABA1, a major constitutive QTL, affects maize root architecture and leaf ABA concentration at different water regimes.

Near-isogenic hybrids (NIHs), developed from crossing maize (Zea mays L.) backcross-derived lines (BDLs) differing for the parental alleles at a major QTL for leaf ABA concentration (L-ABA), were field-tested for 2 years under well-watered and water-stressed conditions. Differences among NIHs for L-ABA and other morpho-physiological traits were not affected by water regimes. On average, the QTL allele for high L-ABA markedly reduced stomatal conductance and root lodging. To elucidate the effects of the QTL on root architecture and L-ABA, root traits of two pairs of BDLs were measured in plants grown in soil columns at three water regimes. Differences among BDLs were not affected by water regimes. Across water regimes, the QTL confirmed its effect on L-ABA and showed a concurrent effect on root angle, branching, number, diameter, and dry weight. Based on these results, it is concluded that the QTL affects root lodging through a constitutive effect on root architecture. In addition, there is speculation that the QTL effects on root traits and L-ABA are probably due to pleiotropy rather than linkage and a model is proposed in which the QTL has a direct effect on root architecture, while indirectly affecting L-ABA.

Mapping QTLs regulating morpho-physiological traits and yield: case studies, shortcomings and perspectives in drought-stressed maize.

Comparative analysis of a number of studies in drought-stressed maize (Zea mays L.) reporting quantitative trait loci (QTLs) for abscisic acid concentration, root characteristics, other morpho-physiological traits (MPTs) and grain yield (GY) reveals their complex genetic basis and the influence of the genetic background and the environment on QTL effects. Chromosome regions (e.g. near umc11 on chromosome 1 and near csu133 on chromosome 2) with QTLs controlling a number of MPTs and GY across populations and conditions of different water supply have been identified. Examples are presented on the use of QTL information to elucidate the genetic and physiological bases of the association among MPTs and GY. The QTL approach allows us to develop hypotheses accounting for these associations which can be further tested by developing near isogenic lines (NILs) differing for the QTL alleles. NILs also allow for a more accurate assessment of the breeding value of MPTs and, in some cases, may allow for the map-based cloning of the gene(s) underlying the QTL. Although QTL analysis is still time-consuming and resource-demanding, its integration with genomics and post-genomics approaches (e.g. transcriptome, proteome and metabolome analyses) will play an increasingly important role for the identification and validation of candidate genes affecting MPTs and GY.

Identification of QTLs for root characteristics in maize grown in hydroponics and analysis of their overlap with QTLs for grain yield in the field at two water regimes.

We investigated the overlap among quantitative trait loci (QTLs) in maize for seminal root traits measured in hydroponics with QTLs for grain yield under well-watered (GY-WW) and water-stressed (GY-WS) field conditions as well as for a drought tolerance index (DTI) computed as GY-WS/GY-WW. In hydroponics, 11, 7, 9, and 10 QTLs were identified for primary root length (R1L), primary root diameter (R1D), primary root weight (R1W), and for the weight of the adventitious seminal roots (R2W), respectively. In the field, 7, 8, and 9 QTLs were identified for GY-WW, GY-WS, and DTI, respectively. Despite the weak correlation of root traits in hydroponics with GY-WW, GY-WS, and DTI, a noticeable overlap between the corresponding QTLs was observed. QTLs for R2W most frequently and consistently overlapped with QTLs for GY-WW, GY-WS, and/or DTI. At four QTL regions, an increase in R2W was positively associated with GY-WW, GY-WS, and/or DTI. A 10 cM interval on chromosome 1 between PGAMCTA205 and php20644 showed the strongest effect on R1L, R1D, R2W, GY-WW, GY-WS, and DTI. These results indicate the feasibility of using hydroponics in maize to identify QTL regions controlling root traits at an early growth stage and also influencing GY in the field. A comparative analysis of the QTL regions herein identified with those described in previous studies investigating root traits in different maize populations revealed a number of QTLs in common.