Sequence variation in 3′UTR region of crtRB1 gene and its effect on β-carotene accumulation in maize kernel

β-carotene fortification of maize has emerged as a potential, long-term and sustainable approach to alleviate vitamin A deficiency in humans. Among the several genes involved in the carotenoid biosynthetic pathway, the 543 bp allele at crtRB1 3′TE (Transposable Element) gene (allele 1, without insertion) is associated with higher β-carotene accumulation. Estimation of β-carotene through high performance liquid chromatography showed that the CIMMYT genotypes with allele 1 had high kernel β-carotene content whereas the Indian inbreds with the same allele had low β-carotene content. To know the reason for this variation, allele 1 of crtRB1 3′TE gene was sequenced from a set of 11 diverse maize inbreds collected from CIMMYT and Indian germplasm. The sequence data of the allele 1 revealed the presence of 13 single nucleotide polymorphisms (SNPs) and 7 insertions and deletions (InDels). Exonic region had two SNPs, intronic region had one SNP and one InDel, whereas 3′-untranslated region (UTR) region of the gene showed 10 SNPs and 6 InDels. Among the several SNPs and InDels, SNP4, SNP13, InDel6 and InDel7 identified in the 3′-UTR region clearly differentiated the high and the low β-carotene genotypes. These 3′-UTR polymorphisms in allele 1 of the crtRB1 3′TE gene could be associated with the variation in kernel β-carotene accumulation by regulating the translation and stability of the mRNA. The SNPs and the InDels associated with higher level of β-carotene will be used as a gene-based marker(s) in selection of genotypes and to develop biofortified maize hybrids to alleviate vitamin A deficiency in humans.     

Single large-scale marker-assisted selection (SLS-MAS)

This paper presents a new approach for plant improvement that interactively combines the use of DNA markers and conventional breeding. This approach involves selecting plants at early generation with a fixed, favorable genetic background at specific loci, conducting a single large-scale marker-assisted selection (SLS-MAS) while maintaining as much as possible the allelic segregation in the rest of the genome. First, the identification of elite lines presenting high allelic complementarity and being outstanding for traits of interest is required to capture favorable alleles from different parental lines. Second, after identification of the most favorable genomic regions for each selected parental line, those lines are intercrossed to develop segregating populations from which plants homozygous for favorable alleles at target loci are selected. One objective of the scheme is to conduct the marker-assisted selection only once, and it requires the selection of a minimum number of plants to maintain sufficient allelic variability at the unselected loci. Therefore, the selection pressure exerted on the segregating population is quite high and the screening of large populations is required to achieve the objectives of the scheme. No selection is applied outside the target genomic regions, to maintain as much as possible the Mendelian allelic segregation among the selected genotypes. After selection with DNA markers, the genetic diversity at un-selected loci may allow breeders to generate new varieties and hybrids through conventional breeding in response to various local needs. Although the single large-scale MAS scheme described here is oriented toward maize and large-scale breeding programs with substantial resources, the flexibility of this scheme would allow breeding programs to develop options compatible with local resources.     

Establishment of introgression libraries in hybrid rye (Secale cereale L.) from an Iranian primitive accession as a new tool for rye breeding and genomics

Genetic diversity of elite breeding material can be increased by introgression of exotic germplasm to ensure long-term selection response. The objective of our study was to develop and characterize the first two rye introgression libraries generated by marker-assisted backcrossing and demonstrate their potential application for improving the baking quality of rye. Starting from a cross between inbred line L2053-N (recurrent parent) and a heterozygous Iranian primitive population Altevogt 14160 (donor) two backcross (BC) and three selfing generations were performed to establish introgression libraries A and B. Amplified fragment length polymorphisms (AFLP markers) and simple sequences repeats (SSRs) were employed to select and characterize candidate introgression lines (pre-ILs) from BC(1) to BC2S3. The two introgression libraries comprise each 40 BC2S3 pre-ILs. For analyzing the phenotypic effects of the exotic donor chromosome segment (DCS) we evaluated the per se performance for pentosan and starch content in replicated field trials at each of four locations in 2005 and 2006. Introgression library A and B cover 74 and 59% of the total donor genome, respectively. The pre-ILs contained mostly two to four homozygous DCS, with a mean length of 12.9 cM (A) and 10.0 cM (B). We detected eight (A) and nine (B) pre-ILs with a significant (P<0.05) higher pentosan content and two pre-ILs (B) with a significant (P<0.05) higher starch content than the elite recurrent parent. Thus, our results indicate that exotic genetic resources in rye carry favorable alleles for baking quality traits, which can be exploited for improving the elite breeding material by marker-assisted selection (MAS). These introgression libraries can substantially foster rye breeding programs and provide a promising opportunity to proceed towards functional genomics.     

Marker-assisted selection for low phytic acid (lpa1-1) with single nucleotide polymorphism marker and amplified fragment length polymorphisms for background selection in a maize backcross breeding programme

The level of phytic acid is difficult to assess in a maize breeding programme, therefore a co-dominant single nucleotide polymorphism (SNP) marker was used to detect the single recessive low phytic acid (lpa1-1) gene in a BC2F1 population developed from a locally adapted tropical normal inbred line (P 16) and CM 32 (lpa1-1 donor). High-resolution melt analysis of the lpa1-1 SNP marker was able to identify 11 homozygous recessive and 17 heterozygote genotypes for the lpa1-1 mutation. The SNP R ² values for the heterozygotes were higher (90.95?C99.59%) than the lpa1-1 recessives (82.81?C99.58%). The selected BC2F1 lines were fingerprinted with six amplified fragment length polymorphism (AFLP) EcoRI/MseI primer combinations to determine the amount of recurrent parent genome present. The 277 AFLP markers were clearly able to differentiate all the BC2F1 lines from each other and the parental controls with a similarity range from 62.12 to 92.15%. It is expected in the BC2 generation to find 87.5% similarity to the recurrent parent, however in this study higher levels of similarity in 13 BC2F1 lines (six heterozygotes and seven homozygous recessive) with 92.15?C83.33% similarity were observed. The use of marker-assisted selection for foreground and background selection greatly increased the efficiency of detection of the homozygous recessive (99.58%) and heterozygous (99.59%) genotypes as well as improving the recovery of the recurrent parent (92.15%) in the BC2F1 generation of the maize backcross breeding programme.     

标记辅助回交育种中所需最小样本容量的近似估计

回交育种是把有利基因从供体亲本向受体亲本转移的一种有效方法,标记辅助选择可加速其进程。为了制定合理的标记辅助选择计划,育种家必须知道所需的后代群体大小。该文提出了一种估算在标记辅助回交育种中同时进行前景选择和背景选择所需群体大小的方法。在假定所需转移的目标基因座与遗传背景之间为相互独立的简化假设下,可以通过将解析方法（针对前景选择）与基于回交亲本图示基因型的模拟方法（针对背景选择）相结合,近似地估计出在每一世代中选到所需基因型的概率,进而估算出在一定概率水平下至少获得一个符合要求的个体所需的最小样本容量,用假想的例子演示了该方法的使用情况。该方法可以很方便地应用于实际的回交育种。     

Genotyping with real-time PCR reveals recessive epistasis between independent QTL conferring resistance to common bacterial blight in dry bean

Resistance to common bacterial blight in common bean is a complex trait that is quantitatively inherited. Combining QTL is the current strategy for improving resistance, but interactions among different QTL are unknown. We examined the interaction between two independent QTL present in dry bean breeding line XAN 159. The QTL were studied in a near isogenic population consisting of 120 BC(6):F(2) plants. Each BC(6):F(2) plant was evaluated for disease reaction at several time points after pathogen inoculation and the dominant SCAR markers linked with QTL on linkage groups B6 (BC420 ~ QTL) and B8 (SU91 ~ QTL) were interpreted as codominant markers using real time PCR assays. This enabled assignment of BC(6):F(2) plants to all nine possible genotypes. Reaction to CBB in BC(6):F(2) plants was characterized by an epistatic interaction between BC420 and SU91 such that: 1) the expression of BC420 was epistatically suppressed by a homozygous recessive su91//su91 genotype; 2) SU91//SU91 and SU91//su91 genotypes conditioned an intermediate disease reaction when homozygous recessive for bc420//bc420; and 3) the highest level of disease resistance was conferred by genotypes with at least a single resistance allele at both QTL (BC420//-; SU91//-). Segregation for resistance among BC(6):F(3) plants derived from BC(6):F(2) plants that were heterozygous for both QTL did not deviate significantly from expected ratios of 9 resistant: 3 moderately resistant: 4 susceptible. This is consistent with a recessive epistatic model of inheritance between two loci. These results indicate breeders will realize greatest gains in resistance to CBB by selecting breeding materials that are fixed for both QTL. This is a first report of a qualitative digenic model of inheritance discerning an interaction between two QTL conditioning disease resistance in plants.     

Identification of agronomically favorable quantitative trait loci alleles from a dent corn inbred Dan232 using advanced backcross QTL analysis and comparison with the F<Subscript>2:3</Subscript> population in popcorn

Specific traits are an important consideration in plant breeding. In popcorn, inferior agronomic traits could be improved using dent or flint corn backcrossed with popcorn. In this study, we used advanced backcross quantitative trait locus (AB-QTL) analysis to identify trait-improving QTL alleles from a dent maize inbred Dan232, and compared the detection of QTL in the BC₂S₁ population with QTL results using F_2:3 families of the same population. Two hundred and twenty BC₂S₁ families developed from a cross between Dan232 and an elite popcorn inbred N04 were evaluated for nine plant traits in replicated field trials under two environments. Using composite interval mapping (CIM), a total of 28 significant QTL were detected, and of these, 23 (82.14%) had favorable alleles contributed by the dent corn parent Dan232. Nine QTL (32.14%) detected in the BC₂S₁ population were also located in or near the same chromosome intervals in the F_2:3 population. All of the favorable QTL alleles from Dan232 could be used in marker-assisted selection (MAS) to improve the respective plant traits in popcorn breeding. In addition, their near isogenic lines (QTL-NILs) could be obtained through selfing or another 1–2 backcross with N04. Also, N04 improved for the studied plant traits could be developed from the BC₂S₁ families used in this study. This study demonstrated that the AB-QTL method can be applied to identify favorable QTL from dent corn inbred in popcorn breeding and, once identified, the alleles could be used in marker-assisted selection to improve the respective plant traits.     

Genetic basis of kernel nutritional traits during maize domestication and improvement

The nutritional traits of maize kernels are important for human and animal nutrition, and these traits have undergone selection to meet the diverse nutritional needs of humans. However, our knowledge of the genetic basis of selecting for kernel nutritional traits is limited. Here, we identified both single and epistatic quantitative trait loci (QTLs) that contributed to the differences of oil and carotenoid traits between maize and teosinte. Over half of teosinte alleles of single QTLs increased the values of the detected oil and carotenoid traits. Based on the pleiotropism or linkage information of the identified single QTLs, we constructed a trait–locus network to help clarify the genetic basis of correlations among oil and carotenoid traits. Furthermore, the selection features and evolutionary trajectories of the genes or loci underlying variations in oil and carotenoid traits revealed that these nutritional traits produced diverse selection events during maize domestication and improvement. To illustrate more, a mutator distance–relative transposable element (TE) in intron 1 of DXS2, which encoded a rate‐limiting enzyme in the methylerythritol phosphate pathway, was identified to increase carotenoid biosynthesis by enhancing DXS2 expression. This TE occurs in the grass teosinte, and has been found to have undergone selection during maize domestication and improvement, and is almost fixed in yellow maize. Our findings not only provide important insights into evolutionary changes in nutritional traits, but also highlight the feasibility of reintroducing back into commercial agricultural germplasm those nutritionally important genes hidden in wild relatives.     

Improvement of hybrid yield by advanced backcross QTL analysis in elite maize

We applied an advanced backcross breeding strategy to identify quantitative trait loci (QTLs) of agronomic importance in a cross between two elite inbreds of maize, RD6502 (Mo17-type recurrent parent) and RD3013 (Iodent donor parent). Two hundred and four BC(2) families were scored at 106 SSR, 15 AFLP, and 38 Heartbreaker (MITE) loci. BC(2) testcrosses (TC) with B73 were phenotyped at six locations in the Midwest and N.Y. We detected four grain yield, six grain moisture, and three plant height QTLs at which the RD3013 allele had a favorable effect ( p < 0.05). All four yield QTLs were selected as target introgressions in the development of BC(3)TC families. As predicted by BC(2)TC analysis, BC(3)TC entries containing introgressions at yld3.1 and yld10.1 significantly outperformed non-carrier entries by 11.1% (15.6 bu/A at one location) and 6.7% (7.1 bu/A averaged across two locations), respectively, in replicated Midwestern trials ( p < 0.05). Detection of yld10.1 effects in the BC(2)TC by spatial analysis (i.e., incomplete block, response surface, autoregressive, moving average or autoregressive moving average), but not by conventional single point analysis or interval mapping, indicated the utility of local environmental control for QTL mapping in unreplicated maize progeny. This work demonstrated that the advanced backcross QTL method can be applied to identify and manipulate useful QTLs in heterotic inbreds of elite maize. Genetic gains by this approach can be coupled with the maintenance and selection of favorable epistatic gene complexes by traditional hybrid breeding for maize improvement.     

Identification of favorable SNP alleles and candidate genes responsible for inflorescence-related traits via GWAS in chrysanthemum

Key message

81 SNPs were identified for three inflorescence-related traits, in which 15 were highly favorable. Two dCAPS markers were developed for future MAS breeding, and six candidate genes were predicted.

Abstract

Chrysanthemum is a leading ornamental species worldwide and demonstrates a wealth of morphological variation. Knowledge about the genetic basis of its phenotypic variation for key horticultural traits can contribute to its effective management and genetic improvement. In this study, we conducted a genome-wide association study (GWAS) based on two years of phenotype data and a set of 92,617 single nucleotide polymorphisms (SNPs) using a panel of 107 diverse cut chrysanthemums to dissect the genetic control of three inflorescence-related traits. A total of 81 SNPs were significantly associated with the three inflorescence-related traits (capitulum diameter, number of ray florets and flowering time) in at least one environment, with an individual allele explaining 22.72–38.67% of the phenotypic variation. Fifteen highly favorable alleles were identified for the three target traits by computing the phenotypic effect values for the stable associations detected in 2 year-long trials at each locus. Dosage pyramiding effects of the highly favorable SNP alleles and significant linear correlations between highly favorable allele numbers and corresponding phenotypic performance were observed. Two highly favorable SNP alleles correlating to flowering time and capitulum diameter were converted to derived cleaved amplified polymorphic sequence (dCAPS) markers to facilitate future breeding. Finally, six putative candidate genes were identified that contribute to flowering time and capitulum diameter. These results serve as a foundation for analyzing the genetic mechanisms underlying important horticultural traits and provide valuable insights into molecular marker-assisted selection (MAS) in chrysanthemum breeding programs.

     

Comparative analysis of response to phenotypic and marker-assisted selection for multiple lateral branching in cucumber (Cucumis sativus L.)

Yield increase in processing cucumber ( Cucumis sativus L.) is positively correlated with an increase in number of fruit-bearing branches. Multiple lateral branching (MLB) is a metric trait controlled by at least five effective factors. Breeding efficacy might be improved through marker-assisted selection (MAS) for MLB. Experiments were designed to independently confirm previously determined linkage of molecular markers (L18-2-H19A SNP, CSWTAAA01 SSR, CSWCT13 SSR, W7-2 RAPD and BC-551 RAPD) to MLB, and to determine their utility in MAS. These markers were present in significantly higher frequency than expected (1, presence:3, absence; p < 0.001) in BC(2) plants selected based on a high MLB phenotype (BC(2)PHE). However, markers that were considered selectively neutral fit the expected segregation of donor parent DNA in BC(2) progeny. Markers linked to MLB were used in MAS of BC(1) and BC(2) plants to produce BC(2)MAS, and BC(3)MAS progeny. Means for MLB in MAS populations were compared with backcross populations developed through phenotypic selection (BC(2)PHE, BC(3)PHE) and by random mating where no selection had been applied (BC(2)RND, BC(3)RND). Statistical analysis showed no significant differences ( p < 0.001) between means of phenotypic (BC(2)PHE = 3.02, BC(3)PHE = 3.29) and marker-aided selection (BC(2)MAS = 3.12, BC(3)MAS = 3.11) for MLB. However, both phenotypic and MAS population means were significantly higher than the random control (BC(2)RND = 2.27, BC(3)RND = 2.41) for MLB. Thus, given the observed response to selection and the rapid life-cycle of cucumber (4 months), markers linked to MLB when used in MAS will most likely be effective tools in cucumber improvement.     

Marker-assisted introgression of favorable alleles at quantitative trait loci between maize elite lines

This article reports the marker-assisted introgression of favorable alleles at three quantitative trait loci (QTL) for earliness and grain yield among maize elite lines. The QTL were originally detected in 1992 by means of ANOVA in a population of 96 recombinant inbred lines (RILs). Introgression started from a selected RIL, which was crossed three times to one of the original parents and then self-fertilized, leading to BC(3)S(1) progenies. Markers were used to assist both foreground and background selection at each generation. At the end of the program, the effect of introgression was assessed phenotypically in agronomic trials, and QTL detection was performed by composite interval mapping among BC(3)S(1) progenies. The marker-assisted introgression proved successful at the genotypic level, as analyzed by precision graphical genotypes, although no emphasis was put on the reduction of linkage drag around QTL. Also, QTL positions were generally sustained in the introgression background. For earliness, the magnitude and sign of the QTL effects were in good agreement with those expected from initial RIL analyses. Conversely, for yield, important discrepancies were observed in the magnitude and sign of the QTL effects observed after introgression, when compared to those expected from initial RIL analyses. These discrepancies are probably due to important genotype-by-environment interactions.     

Genetics and Mechanism of Resistance to Meloidogyne arenaria in Peanut Germplasm

Segregation of resistance to Meloidogyne arenaria in six BC₅F₂ peanut breeding populations was examined in greenhouse tests. Chi-square analysis indicated that segregation of resistance was consistent with resistance being conditioned by a single gene in three breeding populations (TP259-3, TP262-3, and TP271-2), whereas two resistance genes may be present in the breeding populations TP259-2, TP263-2, and TP268-3. Nematode development in clonally propagated lines of resistant individuals of TP262-3 and TP263-2 was compared to that of the susceptible cultivar Florunner. Juvenile nematodes readily penetrated roots of all peanut genotypes, but rate of development was slower (P = 0.05) in the resistant genotypes than in Florunner. Host cell necrosis indicative of a hypersensitive response was not consistently observed in resistant genotypes of either population. Three RFLP loci linked to resistance at distances of 4.2 to 11.0 centiMorgans were identified. Resistant and susceptible alleles for RFLP loci R2430E and R2545E were quite distinct and are useful for identifying individuals homozygous for resistance in segregating populations.     

生长相关分子标记在翘嘴鳜五代中的富集

为进一步了解人工选育对翘嘴鳜生长相关遗传标记的影响作用,研究以翘嘴鳜华康1号的5代选育群体为实验材料,对具有生长相关优势基因型的5个标记的6个位点进行扩增,通过直接测序和聚丙烯酰胺凝胶电泳两种方法分型后,统计其优势基因型个体数目在翘嘴鳜5代中的变化。结果显示,在5代群体中,2个单核苷酸多态性位点和4个微卫星位点优势基因型的数目的分布范围为0-4,从F1到F5代,这6个位点优势基因型的平均值分别为0.36、0.71、0.68、0.77和0.94,优势基因型的平均含量随选育世代的增加呈现递增趋势,从侧面反映了人工选育在一定程度上富集了优良基因。此外,对微卫星位点进行了遗传相似性和遗传距离分析,结果显示,随着选育的进行,后续世代与F1的遗传距离有明显的增大趋势,遗传相似性减小,这符合育种的客观规律。但相邻世代间的遗传距离则逐代减小,遗传相似性逐代增大,说明人工选育将遗传相似性较大的群体保留下来了,这种相似性表现在表型上包括生长快、体重大、体长增加等。F1到F5代处于中度遗传多样性的稳定状态,说明群体还存在选育空间。     

Identification and validation of QTLs for salt tolerance during vegetative growth in tomato by selective genotyping.

The purpose of this study was to identify quantitative trait loci (QTLs) for salt tolerance (ST) during vegetative growth (VG) in tomato by distributional extreme analysis and compare them with the QTLs previously identified for this trait. A BC1 population (N = 792) of a cross between a moderately salt-sensitive Lycopersicon esculentum Mill. breeding line (NC84173, maternal and recurrent parent) and a salt-tolerant L. pimpinellifolium (Jusl.) Mill. accession (LA722) was evaluated for ST in solution cultures containing 700 mM NaCl + 70 mM CaCl2 (electrical conductivity, EC = 64 dS/m and phiw approximately -35.2 bars). Thirty-seven BC1 plants (4.7% of the total) that exhibited the highest ST were selected (referred to as the selected population), grown to maturity in greenhouse pots and self-pollinated to produce BC1S1 progeny seeds. The 37 selected BC1S1 progeny families were evaluated for ST and their average performance was compared with that of the parental BC1 population before selection. A realized heritability of 0.50 was obtained for ST in this population. The 37 selected BC1 plants were subjected to restriction fragment length polymorphism (RFLP) analysis using 115 markers, and marker allele frequencies were determined. Allele frequencies for the same markers were also determined in an unselected BC1 population (N = 119) of the same cross. A trait-based marker analysis (TBA), which measures differences in marker allele frequencies between selected and unselected populations, was used to identify marker-linked QTLs. Five genomic regions were detected on chromosomes 1, 3, 5, 6, and 11 bearing significant QTLs for ST. Except for the QTL on chromosome 3, all QTLs had positive alleles contributed from the salt tolerant parent LA722. Of the five QTLs, three (those on chromosomes 1, 3, and 5) were previously identified for this trait in another study, and thus were validated here. Only one of the major QTLs that was identified in our previous study was not detected here. This high level of conformity between the results of the two studies indicates the genuine nature of the identified QTLs and their potential usefulness for ST breeding using marker-assisted selection (MAS). A few BC1S1 families were identified with most or all of the QTLs and with a ST comparable to that of LA722. These families should be useful for the development of salt tolerant tomato lines via MAS.     

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.     

Parental effects in the inheritance of nonnodulation in peanut

A nonnodulating line (M4-2) and three normal nodulating lines (UF 487A, PI 262090, and Florunner) of peanut (Arachis hypogaea L.) were crossed in full diallel to investigate the inheritance of nodulation. Data from F1, F2, F3, F1BC1, and F2BC1 generations indicated that three genes control nodulation at three independent loci, with nodulation being a product of two genes and inhibited by a third gene when it is dominant and the others are homozygous recessive. A genetic model has been proposed that describes the nonnodulated genotypes as n1n1n2n2N3N3 or n1n1n2n2N3n3 and all other genotypes as normally nodulated except n1n1N2n2N3-, which has reduced nodulation when the n1n2N3 male gamete unites with the n1N2- female gamete or when the n1n2n3 male gamete unites with the n1N2N3 female gamete.