Efficiency gain of marker-assisted backcrossing by sequentially increasing marker densities over generations

Expenses for marker assays are the major costs in marker-assisted backcrossing programs for the transfer of target genes from a donor into the genetic background of a recipient genotype. Our objectives were to (1) investigate the effect of employing sequentially increasing marker densities over backcross generations on the recurrent parent genome (RPG) recovery and the number of marker data points (MDP) required, and (2) determine optimum designs for attaining RPG thresholds of 93–98% with a minimum number of MDP. We simulated the introgression of one dominant target gene for genome models of sugar beet (Beta vulgaris L.) and maize (Zea mays L.) with varying marker distances of 5–80 cM and population sizes of 30–250 plants across BC₁ to BC₃ generations. Employing less dense maps in early backcross generations resulted in savings of over 50% in the number of required MDP compared with using a constant set of markers and was accompanied only by small reductions in the attained RPG values. The optimum designs were characterized by increasing marker densities and increasing population sizes in advanced generations for both genome models. We conclude that increasing simultaneously the marker density and the population size from early to advanced backcross generations results in gene introgression with a minimum number of required MDP.     

Microsatellite Marker Linked to a Leaf Rust Resistance Gene from Triticum monococcum L Transferred to Bread Wheat

Triticum monococcum L, a diploid wheat species closely related to the A genome of cultivated wheats, is highly resistant to leaf rust. A synthetic amphiploid, T. monococcum — T. durum was crossed with T. aestivum cv WL711, highly susceptible to leaf rust. Leaf rust resistant derivatives were selected among backcross generations with the recurrent parent WL711 and cytologically analysed. Chromosome number of the leaf rust resistant BC₁F₃ progenies varied from 39 to 44. Six leaf rust resistant and susceptible bulks from different BC₁F₃ progenies were analysed using 29 wheat microsatellite(WMS) markers already mapped on A genome of bread wheat and found polymorphic among parents. One T. monococcum specific allele of WMS gwm136 locus was found to be closely linked to the leaf rust resistance gene in all the resistant bulks. Differential chromosome number, frequency of univalents and multivalents, however, indicated that the critical T. monococcum chromosome might be present in addition to the A genome chromosomes of wheat, substituted either for the B or D genome chromosome of wheat or translocated to chromosome 1A of wheat in one or the other bulks. The association of the T. monococcum specific allele of WMS gwm136 locus to leaf rust resistance was further confirmed from bulked segregant analysis in BC₂F₁ generation.     

Introgression of heat shock protein (Hsp70 and sHsp) genes into the Malaysian elite chilli variety Kulai (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) through the application of marker-assisted backcrossing (MAB)

Backcrossing together with simple sequence repeat marker strategy was adopted to improve popular Malaysian chilli Kulai (Capsicum annuum L.) for heat tolerance. The use of molecular markers in backcross breeding and selection contributes significantly to overcoming the main drawbacks such as increase linkage drag and time consumption, in the ancient manual breeding approach (conventional), and speeds up the genome recovery of the recurrent parent. The strategy was adopted to introgress heat shock protein gene(s) from AVPP0702 (C. annuum L.), which are heat-tolerant, into the genetic profile of Kulai, a popular high-yielding chilli but which is heat sensitive. The parents were grown on seed trays, and parental screening was carried out with 252 simple sequence repeat markers. The selected parents were crossed and backcrossed to generate F₁ hybrids and backcross generations. Sixty-eight markers appeared to be polymorphic and were used to assess the backcross generation; BC₁F₁, BC₂F₁ and BC₃F₁. The average recipient allele of the selected four BC₁F₁ plants was 80.75% which were used to produce the BC₂F₁ generation. BC₁-P₇ was the best BC₁F₁ plant because it had the highest recovery at 83.40% and was positive to Hsp-linked markers (Hsp70-u2 and AGi42). After three successive generations of backcrossing, the average genome recovery of the recurrent parent in the selected plants in BC₃F₁ was 95.37%. Hsp gene expression analysis was carried out on BC₁F₁, BC₂F₁ and BC₃F₁ selected lines. The Hsp genes were found to be up-regulated when exposed to heat treatment. The pattern of Hsp expression in the backcross generations was similar to that of the donor parent. This confirms the successful introgression of a stress-responsive gene (Hsp) into a Kulai chilli pepper variety. Furthermore, the yield performance viz. plant height, number of fruits, fruit length and weight and total yield of the improved plant were similar with the recurrent parent except that the plant height was significantly lower than the Kulai (recurrent) parent.     

结合SSR标记和STS标记对家蚕无鳞毛翅基因的定位

家蚕突变表型无鳞毛翅(non-lepis wing, nlw)由隐性基因nlw控制。由于家蚕雌性不发生交换, 文章采用有鳞毛翅品系P50和无鳞毛翅品系U06两个品系组配F1代及BC₁回交群体, (U06×P50)×U06和U06×(U06×P50)分别记作BC₁F和BC₁M, 根据已经构建的家蚕SSR分子标记连锁图谱及已经发表的有关序列对nlw基因进行了连锁及定位分析。得到8个与nlw基因连锁的SSR(Simple sequence repeat)标记和1个STS(Sequence-tagged sites)标记。BC₁F群中的所有正常翅个体均表现出与(U06×P50)F₁相同的杂合带型; 而所有无鳞毛个体带型与亲本U06一致, 为纯合型。利用BC₁M群体构建了关于nlw基因的遗传连锁图, 连锁图的遗传距离为125.7 cM, 与nlw基因最近的引物为STS标记cash2p, 图距为11.4 cM。     

Genetic analysis and location of gene for resistance to stripe rust in wheat international differential host Strubes Dickkopf

Strubes Dickkopf is the sixth differential in the world set for wheat stripe (yellow) rust. It is very important to clarify its genetic character of resistance to stripe rust and to develop the molecular markers linked to resistance genes. The NIL Taichung 29*6/Strubes Dickkopf, which was obtained by Strubes Dickkopf as the gene donor and Taichung 29 as the genetic background through backcross breeding, was crossed with the recurrent parent Taichung 29, inbred, and backcrossed to obtain the F₁, F₂ and BC₁ population. The genetic analysis of the cross Taichung 29/(Taichung 29*6/Strubes Dickkopf) was assessed by inoculating the rust race CYR26 at seedling stage. Bulked segregant analysis (BSA) and F₂ segregation analysis were used for detecting polymorphic primers to locate the gene. The resistance of the NIL Taichung 29*6/Strubes Dickkopf to CYR26 was controlled by a single dominant gene, named YrSD. The primer pair Xbarc59 on 5B was linked to YrSD and the genetic distance between Xbarc59 and YrSD was 2.4 cM. The molecular marker Xbarc59 closely linked to the gene YrSD could be used in marker-assisted selection for resistance to stripe rust in wheat breeding programmes.     

Introgression of an imidazolinone-resistance gene from winter wheat (Triticum aestivum L.) into jointed goatgrass (Aegilops cylindrica Host)

Imidazolinone-resistant winter wheat (Triticum aestivum L.) is being commercialized in the USA. This technology allows wheat growers to selectively control jointed goatgrass (Aegilops cylindrica Host), a weed that is especially problematic because of its close genetic relationship with wheat. However, the potential movement of the imidazolinone-resistance gene from winter wheat to jointed goatgrass is a concern. Winter wheat and jointed goatgrass have the D genome in common and can hybridize and backcross under natural field conditions. Since the imidazolinone-resistance gene (Imi1) is located on the D genome, it is possible for resistance to be transferred to jointed goatgrass via hybridization and backcrossing. To study the potential for gene movement, BC₂S₂ plants were produced artificially using imidazolinone-resistant winter wheat (cv. FS-4) as the female parent and a native jointed goatgrass collection as the male recurrent parent. FS-4, the jointed goatgrass collection, and 18 randomly selected BC₂S₂ populations were treated with imazamox. The percentage of survival was 100% for the FS-4, 0% for the jointed goatgrass collection and 6 BC₂S₂ populations, 40% or less for 2 BC₂S₂ populations, and 50% or greater for the remaining 10 BC₂S₂ populations. Chromosome counts in BC₂S₃ plants showed a restoration of the chromosome number of jointed goatgrass, with four out of four plants examined having 28 chromosomes. Sequencing of AHASL1D in BC₂S₃ plants derived from BC₂S₂-6 revealed the sexual transmission of Imi1 from FS-4 to jointed goatgrass. Imi1 conferred resistance to the imidazolinone herbicide imazamox, as shown by the in vitro assay for acetohydroxyacid synthase (AHAS) activity.     

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.     

Resistance QTL confirmed through development of QTL-NILs for barley leaf rust resistance

Near-isogenic lines (NILs) differing with regard to disease QTLs provide valuable material for a more detailed study into the genetic basis of quantitative resistance. Previously obtained information on QTLs that show an effect on leaf rust (Puccinia hordei) in barley was used in a marker-assisted backcross programme. The genome origin in backcross plants was controlled through AFLP marker analysis and graphical genotyping. Plants obtained after the third generation of backcrossing sufficiently resembled the recurrent parent. For one QTL, BC₃S₁ plants were evaluated in a disease test and genotyped. NILs containing the desired QTL in homozygous condition in a recipient background were finally obtained. A disease test and verification of the marker genotype confirmed the identity of the NILs. Simultaneous with the backcross programme a simulation study on efficiency of marker-assisted backcrossing was performed.     

Specific Features of the Nuclear Genome Recombination in Backcross Progenies of Barley–Wheat Hybrids Hordeum vulgare L. (2n = 14) × Triticum aestivum L. (2n = 42)

The backcross progenies of the barley–wheat hybrids Hordeum vulgare L. (2n = 14) × Triticum aestivum L. (2n= 42) and two alloplasmic lines derived from them were studied using microsatellite markers of barley and wheat. The F₁ hybrids and first backcross plants BC₁ contained the genetic material of both cultivated barley and the cultivars of common wheat involved in developing of these hybrid genotypes. The genomes of BC₃, BC₄, and alloplasmic lines contained no microsatellite markers of the cultivated barley, whereas chromosomes of each homeologous group of common wheat were identified. In chromosomes of backcross progenies BC₃, BC₄, and alloplasmic lines yielded by backcrosses of hybrids and various common wheat cultivars, microsatellite markers of the parental wheat cultivars were shown to undergo recombination.     

A BIL Population Derived from G. hirsutum and G. barbadense Provides a Resource for Cotton Genetics and Breeding

To provide a resource for cotton genetics and breeding, an interspecific hybridization between Gossypium hirsutum cv. Emian22 and G. barbadense acc. 3–79 was made. A population of 54 BILs (backcross inbred lines, BC₁F₈) was developed with the aim of transferring G. barbadense genes into G. hirsutum in order to genetically analyze these genes’ function in a G. hirsutum background and create new germplasms for breeding. Preliminary investigation of the morphological traits showed that the BILs had diverse variations in plant architecture, seed size, and fuzz color; the related traits of yield and fiber quality evaluated in 4 environments also showed abundant phenotypic variation. In order to explore the molecular diversity of the BIL population, 446 SSR markers selected at an average genetic distance of 10 cM from our interspecific linkage map were used to genotype the BIL population. A total of 393 polymorphic loci accounting for 84.4% MAF (major allele frequency) > 0.05 and 922 allele loci were detected, and the Shannon diversity index (I) was 0.417 per locus. The average introgression segment length was 16.24 cM, and an average of 29.53 segments were introgressed in each BIL line with an average background recovery of 79.8%. QTL mapping revealed 58 QTL associated with fiber quality and yield traits, and 47 favored alleles derived from the donor parent were discovered. This study demonstrated that the interspecific BIL population was enriched with much phenotypic and molecular variation which could be a resource for cotton genetics and breeding.     

Optimization of linkage mapping strategy and construction of a high-density American lotus linkage map

Background

Lotus is a diploid plant with agricultural, medicinal, and ecological significance. Genetic linkage maps are fundamental resources for genome and genetic study, and also provide molecular markers for breeding in agriculturally important species. Genotyping by sequencing revolutionized genetic mapping, the restriction-site associated DNA sequencing (RADseq) allowed rapid discovery of thousands of SNPs markers, and a crucial aspect of the sequence based mapping strategy is the reference sequences used for marker identification.

Results

We assessed the effectiveness of linkage mapping using three types of references for scoring markers: the unmasked genome, repeat masked genome, and gene models. Overall, the repeat masked genome produced the optimal genetic maps. A high-density genetic map of American lotus was constructed using an F₁ population derived from a cross between Nelumbo nucifera ‘China Antique’ and N. lutea ‘AL1’. A total of 4,098 RADseq markers were used to construct the American lotus ‘AL1’ genetic map, and 147 markers were used to construct the Chinese lotus ‘China Antique’ genetic map. The American lotus map has 9 linkage groups, and spans 494.3 cM, with an average distance of 0.7 cM between adjacent markers. The American lotus map was used to anchor scaffold sequences in the N. nucifera ‘China Antique’ draft genome. 3,603 RADseq markers anchored 234 individual scaffold sequences into 9 megascaffolds spanning 67% of the 804 Mb draft genome.

Conclusions

Among the unmasked genome, repeat masked genome and gene models, the optimal reference sequences to call RADseq markers for map construction is repeat masked genome. This high density genetic map is a valuable resource for genomic research and crop improvement in lotus.     

Genetic analysis and gene mapping of a rice few-tillering mutant in early backcross populations ( Oryza sativa L.)

A rice mutant,G069, characteristic of few tiller numbers, was found in anther culture progeny from theF ₁ hybrid between anindica-japonica cross, Gui630×02428. The mutant has another two major features: delayed tillering development and yellowing apex and margin on the mature leaves. As a donor parent,G069 was further backcrossed with the recurrent parent,02428, for two turns to develop aBC ₂F₂ population. Genetic analysis in theBC ₂F₂ population showed that the traits of few-tillering and yellowing apex and margin on the mature leaves were controlled by one recessive gene. A pool of equally mixed genomic DNA, from few-tillering individual plants inBC ₂F₂, was constructed to screen polymorphism with simple sequence repeat (SSR) markers in comparison with the02428 genome. One SSR marker and three restriction fragment length polymorphism (RFLP) markers were found possibly linked with the recessive gene. By using these markers, the gene of few-tillering was mapped on chromosome 2 between RFLP marker C424 and S13984 with a genetic distance of 2.4 cM and 0.6 cM, respectively. The gene is designatedft1.     

Identification and Validation of Quantitative Trait Loci for Agronomic Traits in Advanced Backcross Breeding Lines Derived from Oryza rufipogon × Oryza sativa Cultivar MR219

A backcross breeding strategy was used to identify quantitative trait loci (QTLs) associated with 14 traits in a BC₂F₂ population derived from a cross between MR219, an indica rice cultivar and an accession of Oryza rufipogon (IRGC 105491). A total of 261 lines were genotyped with 96 microsatellite markers and evaluated for plant morphology, yield components and growth period. The genetic linkage map generated for this population with an average interval size of 16.2?cM, spanning 1,553.4?cM (Kosambi) of the rice genome. Thirty-eight QTLs were identified with composite interval mapping (CIM), whereas simple interval mapping (SIM) resulted in 47 QTLs (LOD >3.0). The O. rufipogon allele was favourable for 59% of QTLs detected through CIM. Of 261 BC₂F₂ families, 26 advanced backcross breeding lines (BC₂F₅) were used for QTL validation. These lines were selected on the basis of the yield traits potentiality in BC₂F₃ and BC₂F₄ generations. The field trial was conducted at three different locations in Malaysia using randomized complete block design with three replications. Trait based marker analysis was done for QTL determination. Twenty-five QTLs were detected in BC₂F₅ generation whereas 29 QTLs were detected in BC₂F₂ generation of the same population. Two QTLs (qPL-1 and qSPL-7) were not considered for validation due to their low R ² values and two QTLs (qPSS-3-2 and qGW-3-2) were not detected in the BC₂F₅ population. Fifteen QTLs showed the beneficial effect to enhance the trait value of the breeding lines. QTL validation aided to select the promising lines for further utilization.     

Genetic analysis and gene mapping of a rice few-tillering mutant in early backcross populations ( Oryza sativa L.)

A rice mutant,G069, characteristic of few tiller numbers, was found in anther culture progeny from theF ₁ hybrid between anindica-japonica cross, Gui630×02428. The mutant has another two major features: delayed tillering development and yellowing apex and margin on the mature leaves. As a donor parent,G069 was further backcrossed with the recurrent parent,02428, for two turns to develop aBC ₂F₂ population. Genetic analysis in theBC ₂F₂ population showed that the traits of few-tillering and yellowing apex and margin on the mature leaves were controlled by one recessive gene. A pool of equally mixed genomic DNA, from few-tillering individual plants inBC ₂F₂, was constructed to screen polymorphism with simple sequence repeat (SSR) markers in comparison with the02428 genome. One SSR marker and three restriction fragment length polymorphism (RFLP) markers were found possibly linked with the recessive gene. By using these markers, the gene of few-tillering was mapped on chromosome 2 between RFLP marker C424 and S13984 with a genetic distance of 2.4 cM and 0.6 cM, respectively. The gene is designatedft1.     

Optimized breeding strategies for multiple trait integration: I. Minimizing linkage drag in single event introgression

From a breeding standpoint, multiple trait integration (MTI) is a four-step process of converting an elite variety/hybrid for value-added traits (e.g. transgenic events) using backcross breeding, ultimately regaining the performance attributes of the target hybrid along with reliable expression of the value-added traits. In the light of the overarching goal of recovering equivalent performance in the finished conversion, this study focuses on the first step of MTI, single event introgression, exploring the feasibility of marker-aided backcross conversion of a target maize hybrid for 15 transgenic events, incorporating eight events into the female hybrid parent and seven into the male parent. Single event introgression is conducted in parallel streams to convert the recurrent parent (RP) for individual events, with the primary objective of minimizing residual non-recurrent parent (NRP) germplasm, especially in the chromosomal proximity to the event (i.e. linkage drag). In keeping with a defined lower limit of 96.66 % overall RP germplasm recovery (i.e. ≤120 cM NRP germplasm given a genome size of 1,788 cM), a breeding goal for each of the 15 single event conversions was developed: <8 cM of residual NRP germplasm across the genome with ~1 cM in the 20 cM region flanking the event. Using computer simulation, we aimed to identify optimal breeding strategies for single event introgression to achieve this breeding goal, measuring efficiency in terms of number of backcross generations required, marker data points needed, and total population size across generations. Various selection schemes classified as three-stage, modified two-stage, and combined selection conducted from BC1 through BC3, BC4, or BC5 were compared. The breeding goal was achieved with a selection scheme involving five generations of marker-aided backcrossing, with BC1 through BC3 selected for the event of interest and minimal linkage drag at population size of 600, and BC4 and BC5 selected for the event of interest and recovery of the RP germplasm across the genome at population size of 400, with selection intensity of 0.01 for all generations. In addition, strategies for choice of donor parent to facilitate conversion efficiency and quality were evaluated. Two essential criteria for choosing an optimal donor parent for a given RP were established: introgression history showing reduction of linkage drag to ~1 cM in the 20 cM region flanking the event and genetic similarity between the RP and potential donor parents. Computer simulation demonstrated that single event conversions with <8 cM residual NRP germplasm can be accomplished by BC5 with no genetic similarity, by BC4 with 30 % genetic similarity, and by BC3 with 86 % genetic similarity using previously converted RPs as event donors. This study indicates that MTI to produce a ‘quality’ 15-event-stacked hybrid conversion is achievable. Furthermore, it lays the groundwork for a comprehensive approach to MTI by outlining a pathway to produce appropriate starting materials with which to proceed with event pyramiding and trait fixation before version testing.     

Two-generation marker-aided backcrossing for rapid conversion of normal maize lines to quality protein maize (QPM)

The low nutritive value of maize endosperm protein is genetically corrected in quality protein maize (QPM), which contains the opaque2 gene along with numerous modifiers for kernel hardness. We report here a two generation marker-based backcross breeding program for incorporation of the opaque2 gene along with phenotypic selection for kernel modification in the background of an early maturing normal maize inbred line, V25. Using the flanking marker distances from opaque2 gene in the cross V25×CML176, optimum population size for the BC₂ generation was computed in such a way that at least one double recombinant could be obtained. Whole genome background selection in the BC₂ generation identified three plants with 93 to 96% recurrent parent genome content. The three BC₂F₂ families derived from marker identified BC₂ individuals were subjected to foreground selection and phenotypic selection for kernel modification. The tryptophan concentration in endosperm protein was significantly enhanced in all the three classes of kernel modification viz., less than 25%, 25–50% and more than 50% opaqueness. BC₂F₃ lines developed from the hard endosperm kernels were evaluated for desirable agronomic and biochemical traits in replicated trials and the best line was chosen to represent the QPM version of V25, with tryptophan concentration of 0.85% in protein. The integrated breeding strategy reported here can be applied to reduce genetic drag as well as the time involved in a conventional line conversion program, and would prove valuable in rapid development of specialty corn germplasm.     

Introgression of genes from Oryza officinalis Well ex Watt to cultivated rice,O. sativa L.

Summary Sterile AC hybrids between cultivated Oryza sativa (AA) and a distant wild species, O. officinalis (CC), were backcross to O. sativa. Most of the BC₁ progenies were allotriploid (AAC), a few were hypotriploid. AAC progenies were again backcrossed to O. sativa. BC₂ progenies consisting of disomic or aneuploid individuals were examined for the presence of O. officinalis traits. Eleven different traits from O. officinalis were identified in these progenies. Segregation data in the subsequent generations suggest that these traits are monogenic in nature. Two of these genes — for resistance to BPH and WBPH — are of value in rice improvement. The extremely low recovery of recombinant progenies is in agreement with the very low amount of pairing between A and C genomes. Because of this restricted recombination, the genotype of the recurrent parent was reconstituted after two backcrosses only. Thus, the BC₂ progenies look remarkably similar to O. sativa. Most of them are stable and fertile and also interfertile with other O. sativa breeding lines. Some of the BPH-and WBPH-resistant progenies are comparable in yield to the best O. sativa parents and are being evaluated as varietal possibilities.     

Genetic Analysis and Physical Mapping of Lk-4(t), a Major Gene Controlling Grain Length in Rice, with a BC2F2 Population

Grain size and shape are important factors affecting grain quality and yield in rice. Mapping, tagging and identification of their related genes can lead us to understand their expression pattern and mechanism network, which is to their control. In this study we mapped a grain length controlling gene named Lk-4(t) with SSR and CAPs markers by screening 800 recessive plants in a BC₂F₂ population derived from a cross of Shuhui527xXiaoli and backcrossed with Xiaoli as the donor parent. The distribution of grain shape parameters and thousand grain weight in F₂ and BC₂F₂ population showed that backcross can diminish most unnecessary variations to identify the target gene more clearly. There were only two grain length phenotypes found among the 3 209 BC₂F₂ plants, long and short, indicating it is a qualitative trait. The frequency distribution for the grain length showed a typical segregation ratio of 3 : 1, suggesting that only one allele was responsible for the variation. By screening the recessive long grain plants with three CAPs markers, P1-EcoR V, P2-Sac I and P3-Mbo I, we tagged the locus on the arm of chromosome 3 near the centromere. Lk-4(t) was located between P1-EcoRV and P2-Sac I, with genetic distance of 0.90 cM and 0.50 cM from the two markers respectively. Mapping of the gene is a foundation for its final identification and function analysis.     

Marker-assisted selection for pyramiding the waxy and opaque-16 genes in maize using cross and backcross schemes

The waxy (wx) gene in maize is associated with higher content of amylopectin in the endosperm and better flavor. The opaque-16 (o16) gene is associated with higher lysine content in the endosperm and better nutritional value. To pyramid the wx and o16 genes, cross and backcross populations were constructed using the o16 line QCL3024 and the two waxy lines, QCL5019 and QCL5008, as parents. The linkage marker umc1141 for the o16 gene and the internal marker phi027 for the wx gene were used to select the target genes. Simple sequence repeat markers covering the whole genome were used for background selection in individual progenies of the backcross population. The grain lysine content was determined using the Acid Orange-12 Dye Binding Lysine method. Qualitative and quantitative analyses of the grain content of amylopectin were performed using the I₂-KI procedure and double-wavelength spectrophotometry, respectively. Four lines of the double recessive genotype wxwxo16o16 were obtained from the F4 generation of the cross population and three lines of the same genotype were obtained from the BC2F4 generation of the backcross population. The lysine content of the pyramid lines was 16–27 and 18–28 % higher than the waxy parents QCL5019 and QCL5008, respectively. The pyramid lines had 61–63 % more amylopectin than the high-lysine parent QCL3024. The three pyramid lines from the backcross population had similar genetic background to the waxy parent QCL5008. Our results are of significance for the improvement of maize quality.     

Development, utilization of introgression lines using a synthetic wheat as donor

A series of introgression lines (ILs) were generated from repeated backcrossing between the exotic hexaploid wheat genotype Am3 and the common wheat genotype Laizhou953. Am3 was synthesized by crossing Triticum carthlicum with Aegilops tauschii and was used as the donor parent in this study, and Laizhou953 was used as the recurrent parent. Two hundred and five SSR markers showing polymorphism between the two parents were used to identify the introgressed Am3 chromosome segments in 97 BC4F3 ILs. The introgressed segments in each line and the length of the introgressed segments were estimated according to the wheat SSR consensus map. The introgressed segments from Am3 in the 97 lines covers 37.7% of the donor genome. The introgressed segments were most found on 2D, 3B, 6B, and 1D with coverage of 59.8, 59.5, 59.1, and 59% of the chromosomes, respectively. None of the 97 lines tested contained chromosome 4D segments introgressed from Am3. Introgressed segments for each of the chromosomes were mapped using the consensus wheat linkage map. Nine agronomic traits from BC4F3 lines were evaluated and the phenotype showed most lines have the tendency to be more similar to the recurrent parent. There were lines showing better agronomic traits than the recurrent parent, which indicated the introgression of favorable alleles from the exotic hexaploid wheat into the elite cultivar Laizhou953. Marker and phenotype data were used to identify quantitative trait loci (QTLs) controlling these nine traits. In total, 38, 33, and 28 putative QTLs were detected for seven of the nine traits in 2003, 2004, and 2005, respectively. Some of these agronomic important QTLs were detected in more than one season. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.