Genetic Diversity and Molecular Evolution of Chinese Waxy Maize Germplasm

Waxy maize (Zea mays L. var. certaina Kulesh), with many excellent characters in terms of starch composition and economic value, has grown in China for a long history and its production has increased dramatically in recent decades. However, the evolution and origin of waxy maize still remains unclear. We studied the genetic diversity of Chinese waxy maize including typical landraces and inbred lines by SSR analysis and the results showed a wide genetic diversity in the Chinese waxy maize germplasm. We analyzed the origin and evolution of waxy maize by sequencing 108 samples, and downloading 52 sequences from GenBank for the waxy locus in a number of accessions from genus Zea. A sharp reduction of nucleotide diversity and significant neutrality tests (Tajima’s D and Fu and Li’s F*) were observed at the waxy locus in Chinese waxy maize but not in nonglutinous maize. Phylogenetic analysis indicated that Chinese waxy maize originated from the cultivated flint maize and most of the modern waxy maize inbred lines showed a distinct independent origin and evolution process compared with the germplasm from Southwest China. The results indicated that an agronomic trait can be quickly improved to meet production demand by selection.     

Molecular evidence for post-domestication selection in the <Emphasis Type="Italic">Waxy</Emphasis> gene of Chinese waxy maize

Waxy maize was first reported in China in 1909 and is mainly used in food production in Asia. The evidence for strong domestication selection in the Waxy locus of rice and a selective sweep around its genomic region make us to wonder whether there has been similar selection in Waxy in glutinous maize. To address this issue, DNA sequences of Waxy, three flanking genes and an unlinked gene (Adh1) of 30 accessions sampled from Chinese waxy maize accessions, including representative landraces and inbred lines, were determined in this study. Sharp reduction of nucleotide diversity and significant neutrality tests (Tajima’s D and Fu and Li’s F*) were observed in the Waxy locus in Chinese waxy maize but not in nonglutinous maize; comparison with the unlinked gene confirmed that this pattern was different to Waxy. Sequence analysis across a 143 kb genomic segment centered on the Waxy locus revealed patterns consistent with a selective sweep in the upstream region of Waxy. The selective sweep detected based on current limited genomic sequences exceeded over 50 kb, indicating strong selection in this or a bigger region. However, No sweep effect was detected in the repetitive downstream region of Waxy. Phylogenetic analysis indicated that Chinese waxy maize was domesticated from the cultivated flint maize (Zea mays ssp. mays) that was introduced from the new world. At least two independent deletions in exon 7 (30 bp) and 10 (15 bp) were identified in the Chinese accessions respectively. These findings demonstrate a similar pattern of domestication selection in the Waxy genomic region in both glutinous maize and rice, suggesting that this pattern in the rise of glutinous phenotype is likely in other cereal crops.     

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.     

Identification of Genetic Differentiation between Waxy and Common Maize by SNP Genotyping

Waxy maize (Zea mays L. var. ceratina) is an important vegetable and economic crop that is thought to have originated from cultivated flint maize and most recently underwent divergence from common maize. In this study, a total of 110 waxy and 110 common maize inbred lines were genotyped with 3072 SNPs to evaluate the genetic diversity, population structure, and linkage disequilibrium decay as well as identify putative loci that are under positive selection. The results revealed abundant genetic diversity in the studied panel and that genetic diversity was much higher in common than in waxy maize germplasms. Principal coordinate analysis and neighbor-joining cluster analysis consistently classified the 220 accessions into two major groups and a mixed group with mixed ancestry. Subpopulation structure in both waxy and common maize sets were associated with the germplasm origin and corresponding heterotic groups. The LD decay distance (1500–2000 kb) in waxy maize was lower than that in common maize. Fourteen candidate loci were identified as under positive selection between waxy and common maize at the 99% confidence level. The information from this study can assist waxy maize breeders by enhancing parental line selection and breeding program design.     

STARCH GRANULE (AMYLOPLAST) DEVELOPMENT IN ENDOSPERM OF SEVERAL ZEA MAYS L. GENOTYPES AFFECTING KERNEL POLYSACCHARIDES

Endosperm cell and starch granule (amyloplast) development of six maize (Zea mays L.) genotypes, normal, amylose-extender (ae), sugary (su), waxy (wx), amylose-extender sugary (ae su), and amylose-extender waxy (ae wx), was compared. Endosperms of all genotypes were indistinguishable at 14 days after pollination. Cells were highly vacuolated and those in the central crown area of the kernel contained small starch granules in close association with the nucleus. Cellular and nuclear enlargement occurred during endosperm development in all genotypes, and major and minor gradients in physiological age of endosperm cells were observed in all kernels. Amyloplast development varied with genotype. Plastid development in normal and wx cells was characterized by an initial starch granule formation followed by granule enlargement to cell maturity. Endosperms homozygous for ae (ae, ae su, and ae wx) developed abnormal plastid-granules. Secondary granule formations preceded development of abnormality in ae and ae su, but not in ae wx endosperms. In contrast to ae and ae su starch granules, ae wx granules were highly birefringent indicating a high degree of crystallinity. In all three ae genotypes, abnormality increased as a function of kernel and physiological cell age. The su mutant had two distinct effects on amyloplast development. First, a mobilization of the initially formed starch, and second a synthesis and accumulation of phytoglycogen and the formation of large rounded plastids. In ae su plastid development, there was a mobilization of the starch initially formed (resulting in irregularly shaped, nonbirefringent granules) but only small amounts of phytoglycogen were produced.     

Wide variability in kernel composition, seed characteristics, and zein profiles among diverse maize inbreds, landraces, and teosinte

All crop species have been domesticated from their wild relatives, and geneticists are just now beginning to understand the consequences of artificial (human) selection on agronomic traits that are relevant today. The primary consequence is a basal loss of diversity across the genome, and an additional reduction in diversity for genes underlying traits targeted by selection. An understanding of attributes of the wild relatives may provide insight into target traits and valuable allelic variants for modern agriculture. This is especially true for maize (Zea mays ssp. mays), where its wild ancestor, teosinte (Z. mays ssp. parviglumis), is so strikingly different than modern maize. One obvious target of selection is the size and composition of the kernel. We evaluated kernel characteristics, kernel composition, and zein profiles for a diverse set of modern inbred lines, teosinte accessions, and landraces, the intermediate between inbreds and teosinte. We found that teosinte has very small seeds, but twice the protein content of landraces and inbred lines. Teosinte has a higher average alpha zein content (nearly 89% of total zeins as compared to 72% for inbred lines and 76% for landraces), and there are many novel alcohol-soluble proteins in teosinte relative to the other two germplasm groups. Nearly every zein protein varied in abundance among the germplasm groups, especially the methionine-rich delta zein protein, and the gamma zeins. Teosinte and landraces harbor phenotypic variation that will facilitate genetic dissection of kernel traits and grain quality, ultimately leading to improvement via traditional plant breeding and/or genetic engineering.     

Increasing Lysine Content of Waxy Maize through Introgression of Opaque-2 and Opaque-16 Genes Using Molecular Assisted and Biochemical Development

The low lysine content of waxy maize cannot meet the nutritional requirements of humans, livestock, or poultry. In the present study, the high-lysine genes o2 and o16 were backcrossed into wx lines using the maize high-lysine inbreds TAIXI19 (o2o2) and QCL3021 (o16o16) as donors and the waxy maize inbred line QCL5019 (wxwx) as a receptor. In the triple-cross F₁, backcross, and inbred generations, the SSR markers phi027 and phi112 within the wx and o2 genes and the SSR marker umc1121 linked to the o16 gene were used for foreground selection. Background selection of the whole-genome SSR markers was performed for the selected individuals. The grain lysine content was determined using the dye-binding lysine method. The waxiness of the grain was determined with the I₂-KI staining and dual-wavelength spectrophotometric analysis. The BC₂F₂ generation included 7 plants of genotype wxwxo2o2O16_, 19 plants of genotype wxwxo16o16O2_, and 3 plants of genotype wxwxo2o2o16o16. In these seeds, the average amylopectin content was 96.67%, 96.87%, and 96.62%, respectively, which is similar to that of QCL5019. The average lysine content was 0.555%, 0.380%, and 0.616%, respectively, representing increases of 75.1%, 19.9%, 94.3%, respectively, over QCL5019. The average genetic background recovery rate of the BC₂F₃ families was 95.3%, 94.3%, 94.2%, respectively. Among these 3 wxwxo2o2O16O16 families, 4 wxwxo2o2O16o16 families, and 3 wxwxo2o2o16o16 families, the longest imported parent donor fragment was 113.35 cM and the shortest fragment was 11.75 cM. No significant differences in lysine content were found between the BC₂F₄ seeds and the BC₂F₃ seeds in these 10 families. This allowed us to increase the lysine content of waxy corn and produce seeds with excellent nutritional characteristics suitable for human consumption, animal feed, and food processing. This may be of significance in the breeding of high-quality corn and in improvement of the nutrition of humans, livestock, and poultry.     

The allotetraploidization of maize

Summary Allotetraploidization is the creation of synthetic allotetraploids. The allotetraploidization of maize can be accomplished by concentrating DPA (differential pairing affinity) factors into stocks by a recurrent selection breeding system. Selection is based on pairing configuration frequencies and altered genetic ratios that reflect DPA. Both an observed decline in the quadrivalent frequency per meiocyte from 8.10 to 7.31 and genetic data disclosing a reduction in the average frequency of recessive waxy (wx wx) pollen from Wx Wx wx wx plants from 17.48% to 13.35%, indicate considerable progress has been made toward allotetraploidization. A simple model for the effect of DPA on chromosome pairing and genetic ratios is presented.Contribution from the Agricultural Research Service, U.S. Department of Agriculture, University of Missouri, Missouri Agricultural Experiment Station, Journal Series No. 9806     

Genetic diversity of maize kernel starch-synthesis genes with SNAPs.

Measuring genetic diversity in populations of a crop species is very important for understanding the genetic structure of and subsequently improving the crop species by genetic manipulation. Single-nucleotide amplified polymorphisms (SNAPs) among and within maize populations of waxy, dent, and sweet corns at 25 single-nucleotide polymorphism (SNP) sites in 6 kernel starch-synthesis genes (sh2, bt2, su1, ae1, wx1, and sh1) were determined. Because of the intensive selection of some favorable alleles in starch-synthesis genes during the breeding process, and the resultant strong linkage disequilibrium (LD), the number of haplotypes in each population was far less than expected. Subsequent phenetic clustering analysis with the SNAPs indicated that the dent, waxy, and sweet corns formed distinct subclusters, except in a few incidences. LD was surveyed among SNAPs of intragenic, intergenic, and intrachromosomal SNPs in whole and subpopulations, which revealed that some SNAPs showed high LD with many other SNAPs, but some SNAPs showed low or no significant LD with others, depending on the subpopulation, indicating that these starch genes have undergone different selection in each subpopulation during the breeding process. Because the starch synthesis genes used in this study are important in maize breeding, the genetic diversity, LD, and accessions having rare SNAP alleles might be valuable in maize improvement programs.     

Mutation loci and intragenic selection marker of the granule-bound starch synthase gene in waxy maize

Four pairs of specific PCR primers have been designed on the basis of the sequence of the granule-bound starch synthase gene (GBSS; dominant non-waxy gene Wx) and used to amplify its homologous sequence from thirteen waxy and two non-waxy inbred lines. Results from electrophoresis indicated that the recessive waxy gene was wx, derived from the dominant non-waxy gene Wx by mutation at its 3′ end. The sequence of the mutated 3′ end was amplified by the TAIL-PCR technique. Sequence alignment showed that the mutation of the wx gene was caused by transposition of the aldehyde dehydrogenase gene rf2. Two pairs of specific primers were designed on the basis of the sequence difference between the dominant gene Wx and its mutated recessive allele wx and used as intragenic selection markers to identify individual plants of genotypes WxWx, Wxwx, and wxwx by PCR amplification from the segregating population of the F₂ generation crossed between waxy and non-waxy inbred lines. Iodine solution staining and starch component assay showed that all the 35 F₂ plants identified as genotype WxWx produced non-waxy kernels of the F₃ generation and that all 33 F₂ plants identified as genotype wxwx produced waxy kernels of the F₃ generation. This result can be used to improve the selection efficiency of waxy maize breeding and for selection of other single genes and major polygenes.     

Analyses of sequence polymorphism and haplotype diversity of LEAFY genes revealed post-domestication selection in the Chinese elite maize inbred lines

Post-domestication selection refers to the artificial selection on the loci controlling important agronomic traits during the process of genetic improvement in a population. The maize genes Zfl1 and Zfl2, duplicate orthologs of Arabidopsis LEAFY, are key regulators in plant branching, inflorescence and flower development, and reproduction. In this study, the full gene sequences of Zfl1 and Zfl2 from 62 Chinese elite inbred lines were amplified to evaluate their nucleotide polymorphisms and haplotype diversities. A total of 254 and 192 variants that included SNPs and indels were identified from the full sequences of Zfl1 and Zfl2, respectively. Although most of the variants were found to be located in the non-coding regions, the polymorphisms of CDS sequences classified Zfl1 into 16 haplotypes encoding 16 different proteins and Zfl2 into 18 haplotypes encoding eight different proteins. The population of Huangzaosi and its derived lines showed statistically significant signals of post-domestication selection on the Zfl1 CDS sequences, as well as lower nucleotide polymorphism and haplotype diversity than the whole set. However, the Zfl2 locus was only selected for in the heterotic group Reid. Further evidence revealed that at least 17 recombination events contributed to the genetic and haplotype diversities at the Zfl1 locus and 16 recombination events at the Zfl2 locus.     

Genetic Analysis of Central Carbon Metabolism Unveils an Amino Acid Substitution That Alters Maize NAD-Dependent Isocitrate Dehydrogenase Activity

Background

Central carbon metabolism (CCM) is a fundamental component of life. The participating genes and enzymes are thought to be structurally and functionally conserved across and within species. Association mapping utilizes a rich history of mutation and recombination to achieve high resolution mapping. Therefore, applying association mapping in maize (Zea mays ssp. mays), the most diverse model crop species, to study the genetics of CCM is a particularly attractive system.

Methodology/Principal Findings

We used a maize diversity panel to test the CCM functional conservation. We found heritable variation in enzyme activity for every enzyme tested. One of these enzymes was the NAD-dependent isocitrate dehydrogenase (IDH, E.C. 1.1.1.41), in which we identified a novel amino-acid substitution in a phylogenetically conserved site. Using candidate gene association mapping, we identified that this non-synonymous polymorphism was associated with IDH activity variation. The proposed mechanism for the IDH activity variation includes additional components regulating protein level. With the comparison of sequences from maize and teosinte (Zea mays ssp. Parviglumis), the maize wild ancestor, we found that some CCM genes had also been targeted for selection during maize domestication.

Conclusions/Significance

Our results demonstrate the efficacy of association mapping for dissecting natural variation in primary metabolic pathways. The considerable genetic diversity observed in maize CCM genes underlies heritable phenotypic variation in enzyme activities and can be useful to identify putative functional sites.     

Genetic dissection of starch paste viscosity characteristics in waxy maize revealed by high-density SNPs in a recombinant inbred line population

Starch paste viscosity properties are widely used as important indicators for quality estimation in waxy maize. To elucidate the genetic basis of paste viscosity characteristics of waxy maize, seven parameters from the rapid visco analyzer (RVA) profile were analyzed for quantitative trait loci (QTLs) in this study, using a recombinant inbred line population derived from a cross between the inbred lines Tongxi5 and Hengbai522. A high-density linkage map was constructed using 2703 bin markers, covering 1876.20 cM of the whole genome with an average genetic distance of 0.73 cM between adjacent bin markers. Seventy-two QTLs were detected for RVA parameters across 3 years, of which 17 could be identified in 2 years, and 6 identified in all 3 years. Eight QTL clusters were observed to be co-associated with two or more RVA parameters. Three major QTLs, qPV4-1, qTV4-1, and qFV5-2, which explained over 10% of the phenotypic variation, were stably mapped to the chromosomes 4 or 5 in all years. Based on functional annotations, two genes were considered as potential candidate genes for the identified major QTLs. The QTLs and candidate genes identified in this study will be useful for further understanding of the genetic architecture of starch paste viscosity characteristics in waxy maize, and may facilitate molecular breeding for grain quality improvement in breeding programs, and simultaneously provide a basis for cloning of the genes underlying these QTLs.     

Genome-Wide Delineation of Natural Variation for Pod Shatter Resistance in Brassica napus

Resistance to pod shattering (shatter resistance) is a target trait for global rapeseed (canola, Brassica napus L.), improvement programs to minimise grain loss in the mature standing crop, and during windrowing and mechanical harvest. We describe the genetic basis of natural variation for shatter resistance in B. napus and show that several quantitative trait loci (QTL) control this trait. To identify loci underlying shatter resistance, we used a novel genotyping-by-sequencing approach DArT-Seq. QTL analysis detected a total of 12 significant QTL on chromosomes A03, A07, A09, C03, C04, C06, and C08; which jointly account for approximately 57% of the genotypic variation in shatter resistance. Through Genome-Wide Association Studies, we show that a large number of loci, including those that are involved in shattering in Arabidopsis, account for variation in shatter resistance in diverse B. napus germplasm. Our results indicate that genetic diversity for shatter resistance genes in B. napus is limited; many of the genes that might control this trait were not included during the natural creation of this species, or were not retained during the domestication and selection process. We speculate that valuable diversity for this trait was lost during the natural creation of B. napus. To improve shatter resistance, breeders will need to target the introduction of useful alleles especially from genotypes of other related species of Brassica, such as those that we have identified.     

Analysis of genetic mapping in a waxy/dent maize RIL population using SSR and SNP markers

A maize genetic linkage map was generated using SSR and SNP markers in a F_7:8 recombinant inbred line (RIL) population derived from a cross of waxy corn (KW7) and dent corn (Mo17). A total of 465 markers, including 459 SSR and 6 SNP markers, were assigned to 10 linkage groups which spanned 2,656.5 cM with an average genetic distance between markers of 5.7 cM, and the number of loci per linkage group ranged from 39 to 55. The SSR (85.4%) and SNP (83.3%) markers showed Mendelian segregation ratios in the RIL population at a 5% significance threshold. In linkage analysis of six SNP loci associated with kernel starch synthesis genes (ae1, bt2, sh1, sh2, su1, and wx1), all six loci were successfully mapped and are closely linked with SSR markers in chromosomes 3 (sh2), 4 (su1 and bt2), 5 (ae1), and 9 (sh1 and wx1). The SSR markers linked with genes in starch synthesis may be utilized in marker assisted breeding programs. The resulting genetic map will be useful in dissection of quantitative traits and the identification of superior QTLs from the waxy hybrid corn. Additionally, these data support further genetic analysis and development of maize breeding programs.