Molecular genetic mapping of a high-lysine mutant gene (<Emphasis Type="Italic">opaque-16</Emphasis>) and the double recessive effect with <Emphasis Type="Italic">opaque-2</Emphasis> in maize

The lysin content in maize endosperm protein is considered to be one of the most important traits for determining the nutritional quality of food and feed. Improving the protein quality of the maize kernel depends principally on finding a mutant with a higher lysine content. Two high-lysine mutant lines with opaque endosperm, QCL3024 and QCL3021, were isolated from a self-cross population derived from Robertsons Mutator stocks. The gene controlling this mutation is temporarily termed opaque-16 (o16). In order to illuminate the genetic locus and effect of the o16 gene, two F_2:3 populations, one developed from a cross between QCL3024 and QCL3010 (a wild type line) and another from a cross between Qi205 (opaque-2 line) and QCL3021, were created, and F₃ seeds from the F₂ plants in the two populations were evaluated for lysine content. The distributions of lysine content and tests for their normality indicate that the lysine content in the two populations is regulated by the major gene of o16 and genes of o2 and o16, respectively. Based on two data sets of the linkage maps of the F₂ plant marker genotypes and the lysine content of F₃ seeds originating from the two F_2:3 populations, the o16 gene was located within 5 cM, at either 3 or 2.2 cM from umc1141 in the interval between umc1121 and umc1141 on the long arm of chromosome 8, depending on the recombination rate in the two populations as determined by composite interval mapping. According to the data of the F_2:3 population constructed from the o2 and o16 lines, the double recessive mutant effect was analyzed. The average lysine content of the F₃ o2o2o16o16 families identified by the umc1066 and umc1141 markers was approximately 30% higher than that of the F₃ o2o2 and o16o16 families, respectively. The lysine content of seven F₃ families among nine F₃ double recessive mutant families showed different increments, with an average increase of some 6% compared with that of the maternal o2 line. The potential application of the o16 mutant for maize high-lysine breeding may be to combine it with the o2 mutant bearing modifier genes, thus obtaining a mutant with much higher lysine content. For the purpose of pyramiding the o16 with o2 genes, the availability of closely linked markers of the o16 and o2 loci will facilitate marker-assisted selection and greatly reduce breeding time and effort.     

A transposable element insertion within ZmGE2 gene is associated with increase in embryo to endosperm ratio in maize

Most of the maize kernel oil is located in the embryo while the majority of starch is located in the endosperm. Maize kernel composition and value are affected significantly by the ratio of the embryo size to the endosperm size; however, the genetic regulation of embryo to endosperm ratio (EER) in maize is unknown. Here we identified ZmGE2 gene, which encodes a cytochrome p450 protein, as a gene associated with EER variation in maize. We first expressed rice Giant Embryo (GE) gene driven by oleosin promoter in maize and detected a 23.2?% reduction in EER in transgenic seeds, demonstrating the existence of evolutionarily conserved mechanisms for EER determination in rice and maize. We next identified maize GE2, a homolog of rice GE sharing 70?% identity in amino sequence, as a candidate based on the similar expression pattern and co-localization with a previously detected QTL for EER. Followed by linkage and association mapping, a 247-bp transposable element (TE) insertion in 3′-untranslated region of ZmGE2 gene was identified to be associated with increase in EER and kernel oil content. Expression level of the favorable ZmGE2 allele containing the 247-bp TE insertion was strongly reduced. In addition, the 247-bp TE insertion site was a selection target during the artificial long-term selection for the high EER trait in a high oil population. This is the first report that demonstrates an association of ZmGE2 with EER variation in maize and identifies ZmGE2 gene as a promising target for manipulation of EER and grain composition by either transgenic approach or molecular breeding in maize.     

Analysis of genetic effects on nutrient quality traits in indica rice

Nine cytoplasmic male-sterile lines and five restorer lines were used in an incomplete diallel cross to analyze seed effects, cytoplasmic effects, and maternal gene effects on nutrient quality traits of indica rice (Oryza sauva L.). The results indicated that nutrient quality traits were controlled by cytoplasmic and maternal effects as well as by seed direct effects. Maternal effects for lysine content (LC), lysine index (LI), and the ratio of lysine content to protein content (RLP) were more important than seed direct effects, while protein content (PC) and protein index (PI) were mainly affected by seed direct effects. Cytoplasmic effects accounted for 2.41–20.80% of the total genetic variation and were significant for all nutrient quality traits. Additive genetic effects were much more important than dominance effects for all of the traits studied, so that selection could be applied for these traits in early generations.     

Characterization of QTL for oil content in maize kernel

Kernel oil content in maize is a complex quantitative trait. Phenotypic variation in kernel oil content can be dissected into its component traits such as oil metabolism and physical characteristics of the kernel, including embryo size and embryo-to-endosperm weight ratio (EEWR). To characterize quantitative trait loci (QTL) for kernel oil content, a recombinant inbred population derived from a cross between normal line B73 and high-oil line By804 was genotyped using 228 molecular markers and phenotyped for kernel oil content and its component traits [embryo oil content, embryo oil concentration, EEWR, embryo volume, embryo width, embryo length, and embryo width-to-length ratio (EWLR)]. A total of 58 QTL were identified for kernel oil content and its component traits in 26 genomic regions across all chromosomes. Eight main-effect QTL were identified for kernel oil content, embryo oil content, embryo oil concentration, EEWR, embryo weight, and EWLR, each accounting for over 10?% of the phenotypic variation in six genomic regions. Over 90?% of QTL identified for kernel oil content co-localized with QTL for component traits, validating their molecular contribution to kernel oil content. On chromosome 1, the QTL that had the largest effect on kernel oil content (qKO1-1) was associated with embryo width; on chromosome 9, the QTL for kernel oil content (qKO9) was related to EEWR (qEEWR9). Embryo oil concentration and embryo width were identified as the most important component traits controlling the second largest QTL for kernel oil content on chromosome 6 (qKO6) and a minor QTL for kernel oil content on chromosome 5 (qKO5-2), respectively. The dissection of kernel oil QTL will facilitate future cloning and/or functional validation of kernel oil content, and help to elucidate the genetic basis of kernel oil content in maize.     

Seed-specific expression of a lysine rich protein sb401 gene significantly increases both lysine and total protein content in maize seeds

The sb401 gene from potato (Solanum berthaultii) encoding a pollen-specific protein with high lysine content was successfully integrated into the genome of maize plants and its expression was correlated with increased levels of lysine and total protein content in maize seeds. A plasmid vector containing the sb401 gene under the control of a maize seed-specific expression storage protein promoter (P19z) was constructed and introduced into maize calli using microprojectile bombardment. The integration of the sb401 gene into the maize genome was confirmed by Southern blot analysis and its expression was confirmed by Western blot analysis. Quantification of lysine and protein content in R1 maize seeds showed that, compared to the non-transgenic maize control, the lysine content increased by 16.1% to 54.8%, and total protein content increased by 11.6% to 39.0%. There was no visible morphological change in vegetative parts and seeds of the transgenic maize plants. Lysine and protein analysis of the transgenic maize grains showed that the levels of lysine and total protein remained high for six continuous generations, indicating that the elevated lysine and total protein levels were heritable. These results indicate that the sb401 gene could be successfully employed in breeding programmes aimed at improving the nutritional value of maize.     

玉米籽粒性状的遗传效应分析

采用二倍体胚和三倍体胚乳种子遗传模型及其分析方法,以5个玉米自交系及其配制的F1,F2,BC1,BC2世代为材料,研究5个玉米种子性状的胚直接效应、胚乳直接效应、母体效应和细胞质效应。分析结果表明,除粒宽外,各性状的遗传同时由细胞质效应和胚、胚乳、母体基因效应所控制,百粒重主要受胚乳和母体效应的影响,粒长的遗传以母体效应为主,粒宽和粒厚以胚乳效应为主。各部位籽粒百粒重的胚乳直接加性效应与母体加性效应的协方差达到显著或极显著水平,其余性状的胚、胚乳直接效应与母体效应间的协方差均不显著,通过母体植株的遗传表现可以对这些性状进行有效的选择。S22 是改良百粒重的优良亲本。 Abstract：The embryo,endosperm and cytoplasm effects of seven seed traits were studied by genetic model for diploid embryo and triploid endosperm plant seeds using five inbreds and their F1, F2, BC1 and BC2 generations. The estimates of genetic variance components indicated that the inheritance of all other kernel traits was controlled by the four effects except kernel width. The 100?kernel weight was mainly controlled by endosperm and maternal effects , and kernel length was controlled by the maternal effects,while endosperm conrrolled kernel width and kernel thickness. Except the significant or highly significant covariances between the endosperm direct additive and maternal additive effects for 100－kernel weight,all other traits between the embryo or endosperm direct effect and the maternal were not significant. So,maize inbreds could be developed by direct selection based on maternal plants for these traits. S22 was the best inbred of the improvement for kernel weight in this study.     

Recessive allelic variations of three microsatellite sites within the<Emphasis Type="Italic">O2</Emphasis> gene in maize

Recessive allelic variations were investigated at 3 microsatellite (SSR) sites within theO2 gene by using 14 inbredo2 lines and a wild-type line in maize. Among the 15 lines, allelic variations were observed at umc1066, phi057, and phi112 sites. Two alleles were found at the umc1066 site—a recessive allele with 2 perfect GCCAGA repeats and a dominant allele with 3 perfect repeats. Three alleles were found at the phi057 site—2 recessive alleles with 3 and 5 perfect GCC repeats, respectively, and another with 4 perfect repeats consistent with a dominant allele. At least 4 alleles exist at the phi112 site—among which 1 recessive allele has a 1-bp deletion, another has a 15-bp deletion, and other has no PCR products compared to the dominant allele; all the alleles have unchanged AG repeats. The phi057 site in exon 6 was identified to be a hypervariable region in the coding sequence of the02 gene, in addition to the 2 hypervariable regions in exon 1 previously reported. The primary mechanisms underlying the variations in repeat numbers and regions flanking the SSR within theO2 gene appear to be unequal crossing over and replication slippage. Furthermore, base substitution of SSR motif can create heteroalleles and modify the repeat number of SSR. The lysine content of kernel in theO2 ando2 lines correlates to a considerable extent with nucleotide variations at the umc1066, phi057, and phi112 sites. Our study suggests that it is best to use the 3 markers together in molecular marker-assisted selection for high-lysine maize materials.     

Kernel lysine content does not increase in some maize opaque2 mutants

The recessive mutant allele of the opaque2 gene (o2) alters the endosperm protein pattern and increases the kernel lysine content of maize (Zea mays L.). In this study, sequencing results showed that the o2 mutant was successfully introgressed into 12 elite normal maize inbred lines by marker assisted selection (MAS). The average genetic similarity between these normal inbred lines and their o2 near-isogenic lines (NILs) was more than 95%. Kernel lysine content increased significantly in most of o2 NILs lines relative to normal elite inbreds, but remained unchanged in the genetic backgrounds Dan598o2 and Liao2345o2. Moreover, the kernel characteristics of these two o2 NILs did not differ from the other inbred lines. The results of lysine content analysis in the F1 hybrids between Liao2345o2 and Dan598o2 and other o2 NILs demonstrated that gene(s) other than opaque2 may control kernel lysine content in these two o2 NILs. The results of zein analysis showed that 22-kD α-zein synthesis was reduced or absent, and the 19-kD α-zein synthesis was greatly reduced compared with the recurrent parents in most o2 NILs except for Dan598o2 and Liao2345o2. Our results indicate that gene(s) other than opaque2 may play more important roles in zein synthesis and kernel lysine content in some maize genetic backgrounds.     

Heterosis × nutrition interaction in Drosophila melanogaster

Summary The relationship between heterozygosity and the expression of heterosis at two different nutrition levels was investigated using Drosophila melanogaster. Average daily egg production and egg hatchability were measured in two parental strains and in F₁, F₂ and reciprocal backcross generations. Heterosis was more pronounced in the poor nutritional conditions. Two electrophoretic markers used to estimate the level of heterozygosity in F₂ and backcrosses revealed an excess of heterozygous genotypes. Quantitative genetic effects (an additive line effect and individual and maternal heterosis) were estimated for both traits in the two environments. Although this model gave a reasonable fit in most cases, some epistatic interaction would have to be invoked in order to explain fully the results.     

Heterosis in the hybridisation between the monoecious and dioecious subspecies ofEcballium elaterium (L.) Richard

Summary By means of a statistical analysis carried out on a number of samples grown in balanced incomplete blocks, respectively corresponding to filial generations which (pure, hybrid or backcross) can be discerned in the hybridisation between the monoecious and dioecious subspecies of the Mediterranean wild cucurbitaceaeEcballium elaterium (L.) Richard, the possible presence of a group of polygenes which modify or regulate the durability of the life of the plants has been investigated, studying especially the most frequent and the most characteristic observable effect of polygenes, the heterosis or hybrid vigour. Experimental results indicates that there is heterosis with respect to the survival of the individual plant. The occurrence of the phenomenon of heterosis is experimental evidence of the presence of a system of polygenes which has influence upon this particular quality of the plant (survival). Every one of these polygenes in the genotype of a subspecies has alleles somehow different from those of another (ofE. elaterium).     

Improving the mineral reserves and protein quality of maize (Zea mays L.) kernels using unique genes

The effects of the maize genes, o ₂ and Mal, on the concentrations of mineral nutrient cations and amino acid levels in mature maize (Zea mays L) kernels of various inbred lines were studied. Previously, the o ₂ gene has been used to improve the protein quality and increase the mineral nutrient content of kernels from some inbred lines. Genotypes possessing the Mal (multiple aleurone layer) gene, contain more than one row of aleurone cells in their kernels and this gene enhances the effect of the o ₂gene on improving kernel protein quality. Incorporating these genes into the maize genome increased accumulation of several mineral nutrients (including Ca, Mg, Zn, Fe, Mn, Zn and Cu) in some of the experimental lines studied. The physiological basis for this increase of mineral nutrients in the kernels is discussed. The effect of the Mal gene on the kernel amino acid composition and protein quality was also examined. Possibly, these genes could be used in combination in breeding programs to improve kernel quality and nutritional value of maize.     

Molecular marker analysis of genes controlling morphological variation in Brassica rapa (syn. campestris)

Construction of a detailed RFLP linkage map of B. rapa (syn. campestris) made it possible, for the first time, to study individual genes controlling quantitative traits in this species. Ninety-five F₂ individuals from a cross of Chinese cabbage cv Michihili by Spring broccoli were analyzed for segregation at 220 RFLP loci and for variation in leaf, stem, and flowering characteristics. The number, location, and magnitude of genes underlying 28 traits were determined by using an interval mapping method. Zero to five putative quantitative trait loci (QTL) were detected for each of the traits examined. There were unequal gene effects on the expression of many traits, and the inheritance patterns of traits ranged from those controlled by a single major gene plus minor genes to those controlled by polygenes with small and similar effects. The effect of marker locus density on detection of QTL was analyzed, and the results showed that the number of QTL detected did not change when the number of marker loci used for QTL mapping was decreased from 220 to 126; however, a further reduction from 126 to 56 caused more than 15% loss of the total QTL detected. The detection of putative minor QTL by removing the masking effects of major QTL was explored.     

Epistasis in maize (Zea mays L.)

Summary Three flint and three dent maize (Zea mays L.) inbred lines, their possible F₁ crosses, F₂ and backcross progenies, and all possible three-way crosses were evaluated in a three-year experiment for yield, ear moisture, and plant height. The purpose was to estimate genetic parameters in European breeding materials from (i) generation means analysis, (ii) diallel analysis of generation means, and (iii) analysis of F₁ and three-way cross hybrids. Method (i) was based on the F-metric model and methods (ii) and (iii) on the Eberhart-Gardner (1966) genetic model; both models extended for heterotic maternal effects.Differences among generation means for yield and plant height were mainly attributable to dominance effects. Epistatic effects were significantly different from zero in a few crosses and considerably reduced heterosis in both traits. Additive x additive and domiance x dominance effects for yield were consistently positive and negative, respectively. Significant maternal effects were established to the advantage of generations with a heterozygous seed parent. In the diallel analysis, mean squares for dominance effects were greater than for additive effects for yield and plant height but smaller for ear moisture. Though significant for yield and plant height, epistatic variation was small compared to additive and dominance variation. Estimates of additive x additive epistasis for yield were significantly negative in 11 of 15 crosses, suggesting that advantageous gene combinations in the lines had been disrupted by recombination in the segregating generations. The analysis of hybrids supported the above findings regarding the analysis of variance. However, the estimates of additive x additive epistasis for yield were considerably smaller and only minimally correlated with those from the diallel analysis. Use of noninbred materials as opposed to materials with different levels of inbreeding is considered the main reason for the discrepancies in the results.     

Heterosis in hybridization between the monoecious and dioecious subspecies of Ecballium elaterium (L.) Rich

Summary Statistical analysis was performed on a set of samples cultivated in balanced, incomplete blocks corresponding, respectively, to filial generations (pure, hybrid or backcross) distinguishable in the hybridization between the monoecious and dioecious subspecies of Ecballium elaterium (L.) Rich. We thus analyzed the possible existence of polygenes that condition the size of the plants, in particular the most observable characteristic effect of the polygenes, i.e., heterosis or hybrid vigor. The experimental results show that heterosis exists with respect to size of the individual plant. The occurrence of this phenomenon provides experimental evidence for the existence of a system of polygenes that affect this property. With respect to each of these polygenes, the genotype of each subspecies contains different alleles. This system of polygenes very likely plays a role in the phenotypic modification which, regarding the sex of the plant itself, has been recognized and confirmed previously.     

Non-additive gene effects in populations under different methods of selection

Summary The genetic parameters of two quantitative traits, 13-day larval weight and pupal weight, in Tribolium populations developed by reciprocal recurrent selection (RRS) and by within-line purebred selection (WLS) were compared each with the other and also with the parameters of the unselected base populations using the genetic model of Carbonell, Nyquist and Bell. The variability for two and three-way crosses of inbred lines derived from companion populations (two strains, breeds, or varieties used for a terminal cross or hybrid) was analyzed into genetic effects: autosomal additivity (^* g), autosomal heterosis (^* s), sex-linked additivity (L), sex-linked heterosis (LL), general maternal (m), specific maternal or reciprocal (r), additive by additive epistasis (aa), and deviations from the model due, among other causes, to higher order epistasis (dev). One series of crosses involved companion populations with diverse origins. For contrast, a second series of crosses involved companion populations originating from a common heterogenous base population. For the heterotic trait larval weight, ^* g and ^* s effects were equally important and accounted for over 50% of the total variation. The aa epistasis contributed another 20% and was followed in importance by higher order epistasis and general maternal effects. For the more highly heritable trait, pupal weight, ^* g effects were most important with ^* s, aa, and m effects having smaller but significant influences. Sex-linked and reciprocal effects were statistically significant for many crosses, but they were relatively unimportant overall. In general, the unselected base populations showed higher ^* g variation than either RRS or WLS populations with the reverse true for ^* s effects. In agreement with theoretical expectations, RRS was more effective than WLS in exploiting ^* s effects. The aa epistatic effects for larval weight were of major importance in the unselected populations, but RRS and WLS did not differ significantly for exploiting superior aa gene combinations. Companion populations with diverse origins revealed significantly larger variation due to ^* g and ^* s effects in crosses than did populations initiated from a common heterogeneous base.Journal Paper No. 11559 from Purdue University Agricultural Experimental Station     

水稻亚种间杂交粒重及加工品质性状的遗传效应分析

用4个广亲和粳型品种和5个籼型品种为材料,按NCⅡ设计配制杂交组合,获得同一环境下的亲本及F_1植株上的籽粒群体(F_2),对其稻谷千粒重、糙米千粒重、出糙率、总精米率以及整精米率等粒重和加工品质性状进行测定,并按胚乳性状遗传模型和混合线性模型的分析方法对籼粳亚种间杂交稻粒重及加工品质性状的遗传效应进行了研究,结果表明:籼粳交籽粒的粒重及各加工品质性状同时受到胚乳直接基因效应、母体基因效应以及微弱的细胞质效应的影响,但其主要受制于母体加性效应,并且存在一定的胚乳杂种优势和母体杂种优势;不同亲本品种对于粒重及加工品质性状的遗传改良具有不同的作用.     

Genetic and heterosis analysis for cooking quality traits of indica rice in different environments

Genetic effects and genotype×environment (GE) interaction effects on the cooking quality traits of indica rice (Oryza sativa L.) were analyzed based on a genetic model for quantitative traits of triploid endosperm in cereal crops. Nine cytoplasmic male-sterile lines as females and 5 restoring lines as males were used in an incomplete diallel cross over 2 years. The cooking quality traits studied were observed to be mainly controlled by genetic effects, but GE interaction effects, especially for amylose content (AC) and alkali spreading score (ASS), were also indicated. Among the genetic effects, seed direct effects and maternal effects were the main components of AC and ASS, respectively; cytoplasmic effects were the main components of gel consistency (GC). Among the GE interaction effects, AC and ASS were mainly affected by maternal interaction effects and GC by direct interaction effects. Additive effects and/or additive interaction effects were the main factors controlling the performance of rice cooking quality traits except for GC which was affected by dominant interaction effects. For AC and GC, there were seed heterosis and/or maternal heterosis. The predicated genetic effects indicated that four parents were better than the others in improving the rice cooking quality traits of the progenies. It was shown that genetic heterosis and GE interaction heterosis were important, especially for amylose content trait in early season indica rice.     

GIANT EMBRYO encodes CYP78A13, required for proper size balance between embryo and endosperm in rice

Among angiosperms there is a high degree of variation in embryo/endosperm size in mature seeds. However, little is known about the molecular mechanism underlying size control between these neighboring tissues. Here we report the rice GIANT EMBRYO (GE) gene that is essential for controlling the size balance. The function of GE in each tissue is distinct, controlling cell size in the embryo and cell death in the endosperm. GE, which encodes CYP78A13, is predominantly expressed in the interfacing tissues of the both embryo and endosperm. GE expression is under negative feedback regulation; endogenous GE expression is upregulated in ge mutants. In contrast to the loss‐of‐function mutant with large embryo and small endosperm, GE overexpression causes a small embryo and enlarged endosperm. A complementation analysis coupled with heterofertilization showed that complementation of ge mutation in either embryo or endosperm failed to restore the wild‐type embryo/endosperm ratio. Thus, embryo and endosperm interact in determining embryo/endosperm size balance. Among genes associated with embryo/endosperm size, REDUCED EMBRYO genes, whose loss‐of‐function causes a phenotype opposite to ge, are revealed to regulate endosperm size upstream of GE. To fully understand the embryo–endosperm size control, the genetic network of the related genes should be elucidated.