Xinhai Li

Joint linkage-linkage disequilibrium mapping is a powerful approach to detecting quantitative trait loci underlying drought tolerance in maize.

This paper describes two joint linkage–linkage disequilibrium (LD) mapping approaches: parallel mapping (independent linkage and LD analysis) and integrated mapping (datasets analyzed in combination). These approaches were achieved using 2,052 single nucleotide polymorphism (SNP) markers, including 659 SNPs developed from drought-response candidate genes, screened across three recombinant inbred line (RIL) populations and 305 diverse inbred lines, with anthesis-silking interval (ASI), an important trait for maize drought tolerance, as the target trait. Mapping efficiency was improved significantly due to increased population size and allele diversity and balanced allele frequencies. Integrated mapping identified 18 additional quantitative trait loci (QTL) not detected by parallel mapping. The use of haplotypes improved mapping efficiency, with the sum of phenotypic variation explained (PVE) increasing from 5.4% to 23.3% for single SNP-based analysis. Integrated mapping with haplotype further improved the mapping efficiency, and the most significant QTL had a PVE of up to 34.7%. Normal allele frequencies for 113 of 277 (40.8%) SNPs with minor allele frequency (<5%) in 305 lines were recovered in three RIL populations, three of which were significantly associated with ASI. The candidate genes identified by two significant haplotype loci included one for a SET domain protein involved in the control of flowering time and the other encoding aldo/keto reductase associated with detoxification pathways that contribute to cellular damage due to environmental stress. Joint linkage–LD mapping is a powerful approach for detecting QTL underlying complex traits, including drought tolerance.

Genome-wide identification of microRNAs in response to low nitrate availability in maize leaves and roots.

Background Nitrate is the major source of nitrogen available for many crop plants and is often the limiting factor for plant growth and agricultural productivity especially for maize. Many studies have been done identifying the transcriptome changes under low nitrate conditions. However, the microRNAs (miRNAs) varied under nitrate limiting conditions in maize has not been reported. MiRNAs play important roles in abiotic stress responses and nutrient deprivation. Methodology/Principal Findings In this study, we used the SmartArray™ and GeneChip® microarray systems to perform a genome-wide search to detect miRNAs responding to the chronic and transient nitrate limiting conditions in maize. Nine miRNA families (miR164, miR169, miR172, miR397, miR398, miR399, miR408, miR528, and miR827) were identified in leaves, and nine miRNA families (miR160, miR167, miR168, miR169, miR319, miR395, miR399, miR408, and miR528) identified in roots. They were verified by real time stem loop RT-PCR, and some with additional time points of nitrate limitation. The miRNAs identified showed overlapping or unique responses to chronic and transient nitrate limitation, as well as tissue specificity. The potential target genes of these miRNAs in maize were identified. The expression of some of these was examined by qRT-PCR. The potential function of these miRNAs in responding to nitrate limitation is described. Conclusions/Significance Genome-wide miRNAs responding to nitrate limiting conditions in maize leaves and roots were identified. This provides an insight into the timing and tissue specificity of the transcriptional regulation to low nitrate availability in maize. The knowledge gained will help understand the important roles miRNAs play in maize responding to a nitrogen limiting environment and eventually develop strategies for the improvement of maize genetics.

Genome-wide association study identifies candidate genes that affect plant height in Chinese elite maize (Zea mays L.) inbred lines.

Background The harvest index for many crops can be improved through introduction of dwarf stature to increase lodging resistance, combined with early maturity. The inbred line Shen5003 has been widely used in maize breeding in China as a key donor line for the dwarf trait. Also, one major quantitative trait locus (QTL) controlling plant height has been identified in bin 5.05–5.06, across several maize bi-parental populations. With the progress of publicly available maize genome sequence, the objective of this work was to identify the candidate genes that affect plant height among Chinese maize inbred lines with genome wide association studies (GWAS). Methods and Findings A total of 284 maize inbred lines were genotyped using over 55,000 evenly spaced SNPs, from which a set of 41,101 SNPs were filtered with stringent quality control for further data analysis. With the population structure controlled in a mixed linear model (MLM) implemented with the software TASSEL, we carried out a genome-wide association study (GWAS) for plant height. A total of 204 SNPs (P≤0.0001) and 105 genomic loci harboring coding regions were identified. Four loci containing genes associated with gibberellin (GA), auxin, and epigenetic pathways may be involved in natural variation that led to a dwarf phenotype in elite maize inbred lines. Among them, a favorable allele for dwarfing on chromosome 5 (SNP PZE-105115518) was also identified in six Shen5003 derivatives. Conclusions The fact that a large number of previously identified dwarf genes are missing from our study highlights the discovery of the consistently significant association of the gene harboring the SNP PZE-105115518 with plant height (P = 8.91e-10) and its confirmation in the Shen5003 introgression lines. Results from this study suggest that, in the maize breeding schema in China, specific alleles were selected, that have played important roles in maize production.

Meta-analysis of constitutive and adaptive QTL for drought tolerance in maize

The response of plants to drought stress is very complex and involves expression of a lot of genes and pathways for diverse mechanisms and interactions with environments. Many quantitative trait loci(QTL) mapping experiments have given heterogeneous results due to use of different genotypes and populations tested in various environments. Our purpose was to identify some important constitutive and adaptive QTL using meta-analysis and to find specific genes and their families for speculating on drought tolerance networks. A total of 239 QTL detected under water-stressed conditions and 160 detected under control conditions from 12 populations tested in 22 experiments were compiled and compared, resulting in identification of 39 consensus QTL under water stress, and 36 under control conditions. Of them, 32 consensus QTL were supposed to be adaptive while others were constitutive QTL. The consensus QTL on chromosomes 1, 2, 3, 5, 6 and 9 were highly overlapped with several different traits and could be identified under multiple environments, most of which were related to traits of high phenotypic variance. Moreover, 48 candidate genes related to stress tolerance were located in silico in these consensus QTL regions what should facilitate the construction of QTL networks and help to understand the mechanisms related to drought tolerance.

An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design.

Precision DNA/gene replacement is a promising genome-editing tool that is highly desirable for molecular engineering and breeding by design. Although the CRISPR/Cas9 system works well as a tool for gene knockout in plants, gene replacement has rarely been reported. Towards this end, we first designed a combinatory dual-sgRNA/Cas9 vector (construct #1) that successfully deleted miRNA gene regions (MIR169a and MIR827a). The deletions were confirmed by PCR and subsequent sequencing, yielding deletion efficiencies of 20% and 24% on MIR169a and MIR827a loci, respectively. We designed a second structure (construct #2) that contains sites homologous to Arabidopsis TERMINAL FLOWER 1 (TFL1) for homology-directed repair (HDR) with regions corresponding to the two sgRNAs on the modified construct #1. The two constructs were co-transformed into Arabidopsis plants to provide both targeted deletion and donor repair for targeted gene replacement by HDR. Four of 500 stably transformed T0 transgenic plants (0.8%) contained replaced fragments. The presence of the expected recombination sites was further confirmed by sequencing. Therefore, we successfully established a gene deletion/replacement system in stably transformed plants that can potentially be utilized to introduce genes of interest for targeted crop improvement.

Inferring Genome Ancestry and Estimating Molecular Relatedness Among 187 Chinese Maize Inbred Lines

The inference of genome ancestry and the estimation of molecular relatedness are of great importance for breeding efficiency and association studies. Seventy SSR loci, evenly distributed in 10 chromosomes, were assayed for polymorphism among 187 commonly used maize (Zea mays L.) inbreds which represent the genetic diversity in China. The identified 290 alleles served as raw data for estimating population structure using the coalescent linked loci, based on the ADMIXTURE model. Population number, K, has been inferred to be between five and seven. Specifying five subpopulations (K = 5) led to a distinct decrease and specifying K to be greater than six resulted in only minimal increases in the likelihood value. Therefore, population number, K, has been inferred into six subpopulations, which are PA, BSSS (includes Reid), PB, Lan (Lancaster Sure Crop), LRC (Luda Reb Cob, a Chinese landrace, and its derivatives), and SPT (Si-ping-tou, a Chinese landrace and its derivatives). The Kullback-Leibler distance of pairwise subpopulation was also inferred as n xp (187 x 6) Q matrices, which gave a detailed percentage of genetic composition of six subpopulations and molecular relatedness of each line. The genome-wide linkage disequilibrium (LD) indicated that the association studies in QTLs and/or candidate genes might avoid nonfunctional and spurious associations, as most of the LD blocks were broken among diverse germplasm. The defined population structure has given us a clear genetic structure of these lines for breeding practice and established a good basis for association analysis.

Comparative LD mapping using single SNPs and haplotypes identifies QTL for plant height and biomass as secondary traits of drought tolerance in maize

Drought often delays developmental events so that plant height and above-ground biomass are reduced, resulting in yield loss due to inadequate photosynthate. In this study, plant height and biomass measured by the Normalized Difference Vegetation Index (NDVI) were used as criteria for drought tolerance. A total of 305 lines representing temperate, tropical and subtropical maize germplasm were genotyped using two single nucleotide polymorphism (SNP) chips each containing 1536 markers, from which 2052 informative SNPs and 386 haplotypes each constructed with two or more SNPs were used for linkage disequilibrium (LD) or association mapping. Single SNP- and haplotype-based LD mapping identified two significant SNPs and three haplotype loci [a total of four quantitative trait loci (QTL)] for plant height under well-watered and water-stressed conditions. For biomass, 32 SNPs and 12 haplotype loci (30 QTL) were identified using NDVIs measured at seven stages under the two water regimes. Some significant SNP and haplotype loci for NDVI were shared by different stages. Comparing significant loci identified by single SNP- and haplotype-based LD mapping, we found that six out of the 14 chromosomal regions defined by haplotype loci each included at least one significant SNP for the same trait. Significant SNP haplotype loci explained much higher phenotypic variation than individual SNPs. Moreover, we found that two significant SNPs (two QTL) and one haplotype locus were shared by plant height and NDVI. The results indicate the power of comparative LD mapping using single SNPs and SNP haplotypes with QTL shared by plant height and biomass as secondary traits for drought tolerance in maize.

Identification of functional genetic variations underlying drought tolerance in maize using SNP markers.

Single nucleotide polymorphism (SNP) is a common form of genetic variation and popularly exists in maize genome. An Illumina GoldenGate assay with 1 536 SNP markers was used to genotype maize inbred lines and identified the functional genetic variations underlying drought tolerance by association analysis. Across 80 lines, 1 006 polymorphic SNPs (65.5% of the total) in the assay with good call quality were used to estimate the pattern of genetic diversity, population structure, and familial relatedness. The analysis showed the best number of fixed subgroups was six, which was consistent with their original sources and results using only simple sequence repeat markers. Pairwise linkage disequilibrium (LD) and association mapping with phenotypic traits investigated under water-stressed and well-watered regimes showed rapid LD decline within 100-500 kb along the physical distance of each chromosome, and that 29 SNPs were associated with at least two phenotypic traits in one or more environments, which were related to drought-tolerant or drought-responsive genes. These drought-tolerant SNPs could be converted into functional markers and then used for maize improvement by marker-assisted selection.

Trends of grain yield and plant traits in Chinese maize cultivars from the 1950s to the 2000s

Retrospective analyses may provide an understanding of unexploited genetic potential and indicate possible pathways for future yield improvement. The objectives of this study were to present maize(Zea mays L.)yield trends and plant traits changes for maize cultivars from the 1950s to the 2000s in China. Trials were conducted at three locations in 2007 and 2008, and at four locations in 2009. Twenty-seven single hybrids, four double-cross hybrids, and four open-pollinated varieties, were grown at three densities at each location each year. 56% of total yield gain was contributed to breeding from 1950 to 2000. New hybrids had more resistance to compound stress. Levels of response of all hybrids to higher-yielding environments were similar, and greater than that of OPVs. All maize cultivars showed morphological changes for all characteristics tested in a volatile manner from 1950 to 2000, except for relatively stable leaf number. ASI decreased and tolerance to root lodging improved, which were enhanced at higher plant densities. There were no trends for other characteristics at higher densities. Shorter maturity, smaller plant size and more tolerance to root and stalk lodging will be required for further yield improvement. Chinese maize yield improvement can benefit from agronomic strategies at higher plant densities.

Association Analysis of the nced and rab28 Genes with Phenotypic Traits Under Water Stress in Maize

As a very complex quantitative trait, drought tolerance has always been suspended with questions at the molecular level. Abscisic acid (ABA) is the main drought-induced hormone that regulates the expression of many genes related to drought tolerance. 9-cis-epoxycarotenoid dioxygenase (NCED) and ABA-responsive gene protein 28 (RAB28) are key enzymes in ABA biosynthesis and regulating drought tolerance induced by ABA under water stress, respectively. In this study, a total of 22 phenotypic traits including morphological traits, grain yield, and its components were investigated under water stress among 196 maize inbred lines majorly collected from five growing zones of China, and they were further genetically sequenced based on nced and rab28 genes and analyzed sequence polymorphism. The phenotypic analysis results showed that ten traits were highly related to variations of drought tolerance. The sequencing results showed low genetic diversity and evidence of neutral selection in both genes and high linkage disequilibrium level among pairwise polymorphic sites. Nucleotide diversity was significantly lower in the coding region than in non-coding regions. By mixed linear model, 13 and 11 polymorphisms of the nced and rab28 genes, respectively, were associating with phenotypic traits under water stress in two different environments. These results will be useful for further functional molecular breeding in improvement of drought tolerance.