The maize PLASTID TERMINAL OXIDASE (PTOX) locus controls the carotenoid content of kernels.

Carotenoids perform a broad range of important functions in humans; therefore, carotenoid biofortification of maize (Zea mays L.), one of the most highly produced cereal crops worldwide, would have a global impact on human health. PLASTID TERMINAL OXIDASE (PTOX) genes play an important role in carotenoid metabolism; however, the possible function of PTOX in carotenoid biosynthesis in maize has not yet been explored. In this study, we characterized the maize PTOX locus by forward- and reverse-genetic analyses. While most higher plant species possess a single copy of the PTOX gene, maize carries two tandemly duplicated copies. Characterization of mutants revealed that disruption of either copy resulted in a carotenoid-deficient phenotype. We identified mutations in the PTOX genes as being causal of the classic maize mutant, albescent1. Remarkably, overexpression of ZmPTOX1 significantly improved the content of carotenoids, especially ß-carotene (provitamin A), which was increased by ~threefold, in maize kernels. Overall, our study shows that maize PTOX locus plays an important role in carotenoid biosynthesis in maize kernels and suggests that fine-tuning the expression of this gene could improve the nutritional value of cereal grains.

Gene pyramiding of ZmGLK36 and ZmGDIα-hel for rough dwarf disease resistance in maize.

Maize rough dwarf disease (MRDD) caused by pathogenic viruses in the genus Fijivirus in the family Reoviridae is one of the most destructive diseases in maize. The pyramiding of effective resistance genes into maize varieties is a potential approach to reduce the damage resulting from the disease. Two major quantitative trait loci (QTLs) (qMrdd2 and qMrdd8) have been previously identified. The resistance genes ZmGLK36 and ZmGDIα-hel have also been cloned with the functional markers Indel-26 and IDP25K, respectively. In this study, ZmGLK36 and ZmGDIα-hel were introgressed to improve MRDD resistance of maize lines (Zheng58, Chang7-2, B73, Mo17, and their derived hybrids Zhengdan958 and B73 × Mo17) via marker-assisted selection (MAS). The converted lines and their derived hybrids, carrying one or two genes, were evaluated for MRDD resistance using artificial inoculation methods. The double-gene pyramiding lines and their derived hybrids exhibited increased resistance to MRDD compared to the monogenic lines and the respective hybrids. The genetic backgrounds of the converted lines were highly similar (90.85-98.58%) to the recurrent parents. In addition, agronomic trait evaluation demonstrated that pyramiding lines with one or two genes and their derived hybrids were not significantly different from the recurrent parents and their hybrids under nonpathogenic stress, including period traits (tasseling, pollen shedding, and silking), yield traits (ear length, grain weight per ear and 100-kernel weight) and quality traits (protein and starch content). There were differences in plant architecture traits between the improved lines and their hybrids. This study illustrated the successful development of gene pyramiding for improving MRDD resistance by advancing the breeding process. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01466-9.

Identification of the coexisting virus-derived siRNA in maize and rice infected by rice black-streaked dwarf virus.

Rice black-streaked dwarf virus is transmitted by small brown planthoppers, which causes maize rough dwarf disease and rice black-streaked dwarf disease. This virus leads to slow growth or death of the host plants. During the co-evolutionary arms race between viruses and plants, virus-derived small interfering RNAs challenge the plant's defense response and inhibit host immunity through the RNA silencing system. However, it is currently unknown if rice black-streaked dwarf virus can produce the same small interfering RNAs to mediate the RNA silencing in different infected species. In this study, four small RNA libraries and four degradome libraries were constructed by extracting total RNAs from the leaves of the maize (Zea mays) inbred line B73 and japonica rice (Oryza sativa) variety Nipponbare exposed to feeding by viruliferous and non-viruliferous small brown planthoppers. We analyzed the characteristics of small RNAs and explored virus-derived small interfering RNAs in small RNA libraries through high-throughput sequencing. On analyzing the characteristics of small RNA, we noted that the size distributions of small RNAs were mainly 24-nt (19.74%-62.00%), whereas those of virus-derived small interfering RNAs were mostly 21-nt (41.06%-41.87%) and 22-nt (39.72%-42.26%). The 5'-terminal nucleotides of virus-derived small interfering RNAs tended to be adenine or uracil. Exploring the distribution of virus-derived small interfering RNAs hot spots on the viral genome segments revealed that the frequency of hot spots in B73 was higher than those in Nipponbare. Meanwhile, hotspots in the S9 and S10 virus genome segments were distributed similarly in both hosts. In addition, the target genes of small RNA were explored by degradome sequencing. Analyses of the regulatory pathway of these target genes unveiled that viral infection affected the ribosome-related target genes in maize and target genes in metabolism and biosynthesis pathways in rice. Here, 562 and 703 virus-derived small interfering RNAs were separately obtained in maize and rice, and 73 virus-derived small interfering RNAs named as co-vsiRNAs were detected in both hosts. Stem-loop PCR and RT-qPCR confirmed that co-vsiRNA 3.1 and co-vsiRNA 3.5 derived from genome segment S3 simultaneously play a role in maize and rice and inhibited host gene expression. The study revealed that rice black-streaked dwarf virus can produce the same small interfering RNAs in different species and provides a new direction for developing the new antiviral strategies.

Pathogenicity Variation in Two Genomes of Cercospora Species Causing Gray Leaf Spot in Maize.

The gray leaf spots caused by Cercospora spp. severely affect the yield and quality of maize. However, the evolutionary relation and pathogenicity variation between species of the Cercospora genus is largely unknown. In this study, we constructed high-quality reference genomes by nanopore sequencing two Cercospora species, namely, C. zeae-maydis and C. zeina, with differing pathogenicity, collected from northeast (Liaoning [LN]) and southeast (Yunnan [YN]) China, respectively. The genome size of C. zeae-maydis-LN is 45.08 Mb, containing 10,839 annotated genes, whereas that of Cercospora zeina-YN is 42.18 Mb, containing 10,867 annotated genes, of which approximately 86.58% are common in the two species. The difference in their genome size is largely attributed to increased long terminal repeat retrotransposons of 3.8 Mb in total length in C. zeae-maydis-LN. There are 41 and 30 carbohydrate-binding gene subfamilies identified in C. zeae-maydis-LN and C. zeina-YN, respectively. A higher number of carbohydrate-binding families found in C. zeae-maydis-LN, and its unique CBM4, CBM37, and CBM66, in particular, may contribute to variation in pathogenicity between the two species, as the carbohydrate-binding genes are known to encode cell wall-degrading enzymes. Moreover, there are 114 and 107 effectors predicted, with 47 and 46 having unique potential pathogenicity in C. zeae-maydis-LN and C. zeina-YN, respectively. Of eight effectors randomly selected for pathogenic testing, five were found to inhibit cell apoptosis induced by Bcl-2-associated X. Taken together, our results provide genomic insights into variation in pathogenicity between C. zeae-maydis and C. zeina. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome-Wide Characterization of the Maize (Zea mays L.) WRKY Transcription Factor Family and Their Responses to Ustilago maydis.

Members of the WRKY transcription factor (TF) family are unique to plants and serve as important regulators of diverse physiological processes, including the ability of plants to manage biotic and abiotic stressors. However, the functions of specific WRKY family members in the context of maize responses to fungal pathogens remain poorly understood, particularly in response to Ustilago maydis (DC.) Corda (U. maydis), which is responsible for the devastating disease known as corn smut. A systematic bioinformatic approach was herein employed for the characterization of the maize WRKY TF family, leading to the identification of 120 ZmWRKY genes encoded on 10 chromosomes. Further structural and phylogenetic analyses of these TFs enabled their classification into seven different subgroups. Segmental duplication was established as a major driver of ZmWRKY family expansion in gene duplication analyses, while the Ka/Ks ratio suggested that these ZmWRKY genes had experienced strong purifying selection. When the transcriptional responses of these genes to pathogen inoculation were evaluated, seven U. maydis-inducible ZmWRKY genes were identified, as validated using a quantitative real-time PCR approach. All seven of these WKRY proteins were subsequently tested using a yeast one-hybrid assay approach, which revealed their ability to directly bind the ZmSWEET4b W-box element, thereby controlling the U. maydis-inducible upregulation of ZmSWEET4b. These results suggest that these WRKY TFs can control sugar transport in the context of fungal infection. Overall, these data offer novel insight into the evolution, transcriptional regulation, and functional characteristics of the maize WRKY family, providing a basis for future research aimed at exploring the mechanisms through which these TFs control host plant responses to common smut and other fungal pathogens.

Evolutionary Analysis of Respiratory Burst Oxidase Homolog (RBOH) Genes in Plants and Characterization of ZmRBOHs.

The respiratory burst oxidase homolog (RBOH), as the key producer of reactive oxygen species (ROS), plays an essential role in plant development. In this study, a bioinformatic analysis was performed on 22 plant species, and 181 RBOH homologues were identified. A typical RBOH family was identified only in terrestrial plants, and the number of RBOHs increased from non-angiosperms to angiosperms. Whole genome duplication (WGD)/segmental duplication played a key role in RBOH gene family expansion. Amino acid numbers of 181 RBOHs ranged from 98 to 1461, and the encoded proteins had molecular weights from 11.1 to 163.6 kDa, respectively. All plant RBOHs contained a conserved NADPH_Ox domain, while some of them lacked the FAD_binding_8 domain. Plant RBOHs were classified into five main subgroups by phylogenetic analysis. Most RBOH members in the same subgroup showed conservation in both motif distribution and gene structure composition. Fifteen ZmRBOHs were identified in maize genome and were positioned in eight maize chromosomes. A total of three pairs of orthologous genes were found in maize, including ZmRBOH6/ZmRBOH8, ZmRBOH4/ZmRBOH10 and ZmRBOH15/ZmRBOH2. A Ka/Ks calculation confirmed that purifying selection was the main driving force in their evolution. ZmRBOHs had typical conserved domains and similar protein structures. cis-element analyses together with the expression profiles of the ZmRBOH genes in various tissues and stages of development suggested that ZmRBOH was involved in distinct biological processes and stress responses. Based on the RNA-Seq data and qRT-PCR analysis, the transcriptional response of ZmRBOH genes was examined under various abiotic stresses, and most of ZmRBOH genes were up-regulated by cold stress. These findings provide valuable information for further revealing the biological roles of ZmRBOH genes in plant development and abiotic stress responses.

Coordinated regulation of starch synthesis in maize endosperm by microRNAs and DNA methylation.

Starch synthesis is an essential feature of crop filling, but knowledge of the molecular mechanisms regulating the expression of starch synthesis genes (SSGs) is currently limited to transcription factors (TFs). Here, we obtained transcriptome, small RNAome, and DNA methylome data from maize (Zea mays) endosperms during multiple developmental stages and established a regulatory network atlas of starch synthesis. Transcriptome analysis showed a sharp transition at 9-10 days after pollination, when genes involved in starch and sucrose metabolism are upregulated and starch accumulates rapidly. Expression pattern analysis established a comprehensive network between SSGs and TFs. During maize endosperm development, the miRNAs with preferential repression of the expression of TFs, particularly the TFs regulating SSG expression, were extensively downregulated. Specifically, ZmMYB138 and ZmMYB115 affected the transcriptional activities of Du1/Wx and Ae1/Bt2 genes at their respective promoter regions. Remarkably, the two TFs were negatively regulated by the copious expression of Zma-miR159k-3p at the post-transcriptional level. This suggests that miRNAs are important regulators of starch synthesis. Moreover, with the exclusion of the TFs, the expression of both SSGs and miRNAs was globally regulated by DNA methylation. Altogether, the present results (i) establish the regulatory functions of miRNAs and DNA methylation in starch synthesis and (ii) indicate that DNA methylation functions as a master switch.

Using the dominant mutation gene Ae1-5180 (amylose extender) to develop high-amylose maize.

Maize amylose is a type of high value-added starch used for medical, food, and chemical applications. Mutations in the starch branching enzyme (SBEIIb), with recessive ae (amylose extender) and dominant Ae1-5180 alleles, are the primary way to improve maize endosperm amylose content (AC). However, studies on Ae1-5180 mutation are scarce, and its roles in starch synthesis and breeding potential are unclear. We found that the AC of the Ae1-5180 mutant was 47.23%, and its kernels were tarnished and glassy and are easily distinguished from those of the wild type (WT), indicating that the dominant mutant has the classical characteristics of the ae mutant. Starch granules of Ae1-5180 became smaller, and higher in amount with irregular shape. The degree of amylopectin polymerisation changed to induce an increase in starch thermal stability. Compared with WT, the activity of granule-bound starch synthase and starch synthase was higher in early stages and lower in later stages, and other starch synthesis enzymes decreased during kernel development in the Ae1-5180 mutant. We successfully developed a marker (mu406) for the assisted selection of 17 Ae1-5180 near isogenic lines (NILs) according to the position of insertion of the Mu1 transposon in the SBEIIb promoter of Ae1-5180. JH214/Ae1-5180, CANS-1/Ae1-5180, CA240/Ae1-5180, and Z1698/Ae1-5180 have high breeding application potential with their higher AC (> 40%) and their 100-kernel weight decreased to < 25% compared to respective recurrent parents. Therefore, using the dominant Ae1-5180 mutant as a donor can detect the kernel phenotype and AC of Ae1-5180-NILs in advance, thereby accelerating the high-amylose breeding process. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01323-7.

Identification and Fine-Mapping of a Novel QTL, qMrdd2, That Confers Resistance to Maize Rough Dwarf Disease.

Maize rough dwarf disease (MRDD) is caused by a virus and seriously affects maize quality and yield worldwide. MRDD can be most effectively controlled with disease-resistant hybrids of corn. Here, MRDD-resistant (Qi319) and -susceptible (Ye478) parental inbred maize lines and their 314 recombinant inbred lines (RILs) that were derived from a cross between them were evaluated across three environments. A stable resistance QTL, qMrdd2, was identified and mapped using best linear unbiased prediction (BLUP) values to a 0.55-Mb region between the markers MK807 and MK811 on chromosome 2 (B73 RefGen_v3) and was found to explain 8.6 to 11.0% of the total phenotypic variance in MRDD resistance. We validated the effect of qMrdd2 using a chromosome segment substitution line (CSSL) that was derived from a cross between maize inbred Qi319 as the MRDD resistance donor and Ye478 as the recipient. Disease severity index of the CSSL haplotype II harboring qMrdd2 was significantly lower than that of the susceptible parent Ye478. Subsequently, we fine-mapped qMrdd2 to a 315-kb region flanked by the markers RD81 and RD87, thus testing recombinant-derived progeny using selfed backcrossed families. In this study, we identified a novel QTL for MRDD resistance by combining the RIL and CSSL populations, thus providing important genetic information that can be used for breeding MRDD-resistant varieties of maize.

Marker-assisted selection of qMrdd8 to improve maize resistance to rough dwarf disease.

Maize rough dwarf disease (MRDD) is caused by viruses in the Fijivirus genus in the family Reoviridae. MRDD resistance can be improved by a combination of marker-assisted selection (MAS) and conventional breeding strategies. In our previous study, we fine-mapped a major QTL qMrdd8 and developed the functional Indel marker IDP25K. In the present study, qMrdd8 from the donor parent X178 was introgressed into elite inbred lines derived from the three corn heterotic groups using multi-generation backcrossing and MAS. Recipient lines included Huangzao4, Chang7-2, Ye478, Zheng58, Zhonghuang68, B73, and Ji846. Markers used for foreground selection included IDRQ4, IDRQ47, IDP25K, and IDP27K. Background selection was carried out in the BC3 or BC4 using 107 SSR markers to select lines with the highest rate of recovery of the particular recurrent parent genome. Plants from BC4F2 and BC3F2 that carried the shortest qMrdd8 interval from X178 and those with the highest rate of recovery of the recurrent parent genome were then selected to create converted homozygous inbred lines. In 2017, seven converted inbred lines and five hybrids exhibited enhanced resistance to MRDD, while other agronomic traits were not affected under nonpathogenic stress conditions. Thus, the MRDD resistance allele at the qMrdd8 locus, or IDP25K, should be valuable for maize breeding programs in China.

Discovery and Fine Mapping of qSCR6.01, a Novel Major QTL Conferring Southern Rust Resistance in Maize.

Southern corn rust (SCR), an airborne disease caused by Puccinia polysora, can severely reduce the yield of maize (Zea mays L.). Using recombinant inbred lines (RILs) derived from a cross between susceptible inbred line Ye478 and resistant Qi319 in combination with their high-density genetic map, we located five quantitative trait loci (QTLs) against SCR, designated as qSCR3.04, qSCR5.07, qSCR6.01, qSCR9.03, and qSCR10.01, on chromosomes 3, 5, 6, 9, and 10, respectively. Each QTL could explain 2.84 to 24.15% of the total phenotypic variation. qSCR6.01, detected on chromosome 6, with the highest effect value, accounting for 17.99, 23.47, and 24.15% of total phenotypic variation in two environments and best linear unbiased prediction, was a stably major resistance QTL. The common confidence interval for qSCR6.01 was 2.95 Mb based on the B73 RefGen_v3 sequence. The chromosome segment substitution lines (CSSLs) constructed with Qi319 as the donor parent and Ye478 as the recurrent parent were used to further verify qSCR6.01 resistance to SCR. The line CL183 harboring introgressed qSCR6.01 showed obvious resistance to SCR that was distinctly different from that of Ye478 (P = 0.0038). Further mapping of qSCR6.01 revealed that the resistance QTL was linked to insertion-deletion markers Y6q77 and Y6q79, with physical locations of 77.6 and 79.6 Mb, respectively, on chromosome 6. Different from previous major genes or QTLs against SCR on chromosome 10, qSCR6.01 was a newly identified major QTL resistance to SCR on chromosome 6 for the first time. Using RIL and CSSL populations in combination, the SCR-resistance QTL research can be dissected effectively, which provided important gene resource and genetic information for breeding resistant varieties.

Integrated transcriptome, small RNA, and degradome analysis reveals the complex network regulating starch biosynthesis in maize.

BACKGROUND: Starch biosynthesis in endosperm is a key process influencing grain yield and quality in maize. Although a number of starch biosynthetic genes have been well characterized, the mechanisms by which the expression of these genes is regulated, especially in regard to microRNAs (miRNAs), remain largely unclear. RESULTS: Sequence data for small RNAs, degradome, and transcriptome of maize endosperm at 15 and 25 d after pollination (DAP) from inbred lines Mo17 and Ji419, which exhibit distinct starch content and starch granule structure, revealed the mediation of starch biosynthetic pathways by miRNAs. Transcriptome analysis of these two lines indicated that 33 of 40 starch biosynthetic genes were differentially expressed, of which 12 were up-regulated in Ji419 at 15 DAP, one was up-regulated in Ji419 at 25 DAP, 14 were up-regulated in Ji419 at both 15 and 25 DAP, one was down-regulated in Ji419 at 15 DAP, two were down-regulated in Ji419 at 25 DAP, and three were up-regulated in Ji419 at 15 DAP and down-regulated in Ji419 at 25 DAP, compared with Mo17. Through combined analyses of small RNA and degradome sequences, 22 differentially expressed miRNAs were identified, including 14 known and eight previously unknown miRNAs that could target 35 genes. Furthermore, a complex co-expression regulatory network was constructed, in which 19 miRNAs could modulate starch biosynthesis in endosperm by tuning the expression of 19 target genes. Moreover, the potential operation of four miRNA-mediated pathways involving transcription factors, miR169a-NF-YA1-GBSSI/SSIIIa and miR169o-GATA9-SSIIIa/SBEIIb, was validated via analyses of expression pattern, transient transformation assays, and transactivation assays. CONCLUSION: Our results suggest that miRNAs play a critical role in starch biosynthesis in endosperm, and that miRNA-mediated networks could modulate starch biosynthesis in this tissue. These results have provided important insights into the molecular mechanism of starch biosynthesis in developing maize endosperm.

Natural variation in the THICK TASSEL DWARF1 (TD1) gene in the regulation of maize (Zea mays L.) ear-related traits.

THICK TASSEL DWARF1 (TD1) is a critical gene that negatively modulates meristem size during maize inflorescence development and may also regulate ear-related traits. In the present study, the whole genomic DNA sequences and the promoter regions of TD1 were analyzed in 165 diverse maize inbred lines. Polymorphism analysis identified 39 SNPs and five InDels in the genic region of TD1 and allowed 23 haplotypes to be classified. Among these sites, eight SNPs and one InDel were significantly associated with kernel number (KN) (p < 0.05), seven SNPs and one InDel were significantly associated with kernel number per row (KNPR) (p < 0.05), and three SNPs were significantly associated with kernel row number (KRN) (p < 0.05). In addition, 21 SNPs and 14 InDels were identified in the promoter regions of TD1, and two SNPs and seven InDels of these sites were found to be significantly associated with KRN (p < 0.05). The results denote that Hap_7 was the favorable haplotype for both KN and KNPR, and Hap_2 was the elite haplotype for KRN. These favorable haplotypes could be utilized in molecular marker-assisted selection (MAS) to improve KN, KNPR, or KRN, and thereby further increase grain yield in maize breeding programs.

Differences in Molecular Characteristics of Segment 8 in Rice black-streaked dwarf virus and Southern rice black-streaked dwarf virus.

Rice black-streaked dwarf virus (RBSDV) and Southern rice black-streaked dwarf virus (SRBSDV) cause maize rough dwarf disease (MRDD) and rice black-streaked dwarf disease (RBSDD) in China. RBSDV segment 8 (S8) contains the only deletion mutation in the genomes of these viruses, which are both members of the genus Fijivirus. To illuminate the molecular differences between the RBSDV and SRBSDV genomes and better understand the evolution of these viruses, and to determine which virus is specifically associated with MRDD and RBSDD in each region, S8 was analyzed in 66 virus isolates collected from 10 geographic locations in China and 14 S8 sequences obtained from the National Center for Biotechnology Information GenBank. Phylogenetic analysis showed that the pathogen associated with MRDD and RBSDD in the Yellow and Huai River valleys was RBSDV, whereas the pathogen associated with these diseases in Sanya was SRBSDV. Codon usage bias in S8 differed significantly between RBSDV and SRBSDV, as indicated by effective number of codons used by a gene (Nc) and GC values, Nc plots, and variation explained by the first axis in correspondence analysis. The nucleotide identities among these 66 RBSDV and SRBSDV isolates ranged from 66.2 to 68.2%, and were considerably lower than the nucleotide identities within RBSDV (from 94.1 to 99.9%) or SRBSDV (from 93.9 to 100%) isolates. Most S8 polymorphisms were identified in the region from 1,000 to 1,200 bp in RBSDV and in the region from 500 to 700 bp in SRBSDV. The difference in the lengths of RBSDV (1,936 bp) and SRBSDV (1,928 bp) was due to an 8-bp deletion in the 3'-untranslated region of SRBSDV. Six recombination events were detected in S8 in RBSDV and two recombination events were detected in S8 in SRBSDV. Recombination breakpoints were found within the region containing the deletion mutation in nine isolates. However, no recombination events were detected between RBSDV and SRBSDV. Both of these viruses were under negative and purifying selection, although the ratio of nonsynonymous mutations to synonymous mutations (Ka/Ks) for RBSDV S8 (0.0530) was not significantly lower than that of SRBSDV S8 (0.0823, P = 0.1550). We found that SRBSDV was more highly genetically differentiated (product of effective population size and the migration rate among populations < 1; values for the among-populations component of genetic variation or normalized variation > 0.33; and P values of the sequence statistic, the rank statistic, and the nearest-neighbor statistic < 0.01) than RBSDV. However, gene flow between RBSDV and SRBSDV was not frequent.

Analysis of the genetic architecture of maize ear and grain morphological traits by combined linkage and association mapping.

KEY MESSAGE: Using combined linkage and association mapping, 26 stable QTL and six stable SNPs were detected across multiple environments for eight ear and grain morphological traits in maize. One QTL, PKS2, might play an important role in maize yield improvement. In the present study, one bi-parental population and an association panel were used to identify quantitative trait loci (QTL) for eight ear and grain morphological traits. A total of 108 QTL related to these traits were detected across four environments using an ultra-high density bin map constructed using recombinant inbred lines (RILs) derived from a cross between Ye478 and Qi319, and 26 QTL were identified in more than two environments. Furthermore, 64 single nucleotide polymorphisms (SNPs) were found to be significantly associated with the eight ear and grain morphological traits (-log10(P) > 4) in an association panel of 240 maize inbred lines. Combining the two mapping populations, a total of 17 pleiotropic QTL/SNPs (pQTL/SNPs) were associated with various traits across multiple environments. PKS2, a stable locus influencing kernel shape identified on chromosome 2 in a genome-wide association study (GWAS), was within the QTL confidence interval defined by the RILs. The candidate region harbored a short 13-Kb LD block encompassing four SNPs (SYN11386, PHM14783.16, SYN11392, and SYN11378). In the association panel, 13 lines derived from the hybrid PI78599 possessed the same allele as Qi319 at the PHM14783.16 (GG) locus, with an average value of 0.21 for KS, significantly lower than that of the 34 lines derived from Ye478 that carried a different allele (0.25, P < 0.05). Therefore, further fine mapping of PKS2 will provide valuable information for understanding the genetic components of grain yield and improving molecular marker-assisted selection (MAS) in maize.

Rice black-streaked dwarf virus Genome in China: Diversification, Phylogeny, and Selection.

Rice black-streaked dwarf virus (RBSDV), a Fijivirus, causes maize rough dwarf disease and rice black-streaked dwarf disease in the summer maize-growing regions of the Yellow and Huai rivers, respectively, in China. Nevertheless, the diversification and selection of the entire genome from S1 to S10 have not been illuminated. Molecular variation, evolution, conserved regions, and other genomic properties were analyzed in 21 RBSDV isolates from maize (Zea mays L.) and rice (Oryza sativa) hosts sampled from nine geographic locations in China. Low codon adaptation index values ranging from 0.1878 to 0.2918 indicated a low degree of codon-usage bias and low potential expression for all 13 RBSDV open reading frames (ORFs). ORF9-2 showed a stronger effect of codon usage bias than did other ORFs, as the majority of points for this ORF lay close to the standard curve in the Nc plot (the effective number of codons [Nc] versus the frequency of G+C at synonymous third-base positions [GC3]). A 9-bp deletion mutation was detected in the RBSDV genome in the 3' UTR of S8. Nucleotide diversity analysis indicated that the structural proteins of RBSDV, such as S2 and S4, were all more conserved than nonstructural proteins such as S9. Nucleotide diversity (π) was highest among S9 sequences (0.0656), and was significantly higher than among S4 sequences (0.0225, P < 0.01). The number of conserved regions among the 10 segments varied substantially. The highest number of conserved regions (5) was found in S5, whereas no conserved regions were identified in S9. Nucleotide diversity and the number of conserved regions were independent of the lengths of segments. Nucleotide diversity was also not correlated with the number of conserved regions in segments. Ten recombination events in 21 isolates were found in seven segments with breakpoint positions in UTRs, intergenic spacer regions, and gene coding regions. The number of recombination events was also independent of the lengths of segments. RBSDV isolates from China could be phylogenetically classified into two groups using either 10 segment sequences or the concatenated sequence of S1 through S10, regardless of host or geographical location. The phylogenetic tree generated from pairwise nucleotide identities of individual RBSDV segments such as S9 and S3, with nucleotide identity values of 93.74% and 95.86%, respectively, is similar to the tree constructed from the concatenated sequences of the entire RBSDV genome. The 13 RBSDV ORFs were under negative and purifying selection (Ka/Ks < 1). ORF5-2 was under the greatest selection pressure; however, ORF2, which encodes the core protein of RBSDV, was under the lowest selection pressure.

An evolutionarily conserved gene, FUWA, plays a role in determining panicle architecture, grain shape and grain weight in rice.

Plant breeding relies on creation of novel allelic combinations for desired traits. Identification and utilization of beneficial alleles, rare alleles and evolutionarily conserved genes in the germplasm (referred to as 'hidden' genes) provide an effective approach to achieve this goal. Here we show that a chemically induced null mutation in an evolutionarily conserved gene, FUWA, alters multiple important agronomic traits in rice, including panicle architecture, grain shape and grain weight. FUWA encodes an NHL domain-containing protein, with preferential expression in the root meristem, shoot apical meristem and inflorescences, where it restricts excessive cell division. Sequence analysis revealed that FUWA has undergone a bottleneck effect, and become fixed in landraces and modern cultivars during domestication and breeding. We further confirm a highly conserved role of FUWA homologs in determining panicle architecture and grain development in rice, maize and sorghum through genetic transformation. Strikingly, knockdown of the FUWA transcription level by RNA interference results in an erect panicle and increased grain size in both indica and japonica genetic backgrounds. This study illustrates an approach to create new germplasm with improved agronomic traits for crop breeding by tapping into evolutionary conserved genes.

Genetic architecture of the maize kernel row number revealed by combining QTL mapping using a high-density genetic map and bulked segregant RNA sequencing.

BACKGROUND: The maize kernel row number (KRN) is a key component that contributes to grain yield and has high broad-sense heritability (H 2 ). Quantitative trait locus/loci (QTL) mapping using a high-density genetic map is a powerful approach to detecting loci that are responsible for traits of interest. Bulked segregant ribonucleic acid (RNA) sequencing (BSR-seq) is another rapid and cost-effective strategy to identify QTL. Combining QTL mapping using a high-density genetic map and BSR-seq may dissect comprehensively the genetic architecture underlying the maize KRN. RESULTS: A panel of 300 F2 individuals derived from inbred lines abe2 and B73 were genotyped using the specific-locus amplified fragment sequencing (SLAF-seq) method. A total of 4,579 high-quality polymorphic SLAF markers were obtained and used to construct a high-density genetic map with a total length of 2,123 centimorgan (cM) and an average distance between adjacent markers of 0.46 cM. Combining the genetic map and KRN of F2 individuals, four QTL (qKRN1, qKRN2, qKRN5, and qKRN8-1) were identified on chromosomes 1, 2, 5, and 8, respectively. The physical intervals of these four QTL ranged from 4.36 Mb for qKRN8-1 to 7.11 Mb for qKRN1 with an average value of 6.08 Mb. Based on high-throughput sequencing of two RNA pools bulked from leaves of plants with extremely high and low KRNs, two QTL were detected on chromosome 8 in the 10-25 Mb (BSR_QTL1) and 60-150 Mb (BSR_QTL2) intervals. According to the physical positions of these QTL, qKRN8-1 was included by BSR_QTL2. In addition, qKRN8-1 was validated using QTL mapping with a recombinant inbred lines population that was derived from inbred lines abe2 and B73. CONCLUSIONS: In this study, we proved that combining QTL mapping using a high-density genetic map and BSR-seq is a powerful and cost-effective approach to comprehensively revealing genetic architecture underlying traits of interest. The QTL for the KRN detected in this study, especially qKRN8-1, can be used for performing fine mapping experiments and marker-assisted selection in maize breeding.

Genetic dissection of maize plant architecture with an ultra-high density bin map based on recombinant inbred lines.

BACKGROUND: Plant architecture attributes, such as plant height, ear height, and internode number, have played an important role in the historical increases in grain yield, lodging resistance, and biomass in maize (Zea mays L.). Analyzing the genetic basis of variation in plant architecture using high density QTL mapping will be of benefit for the breeding of maize for many traits. However, the low density of molecular markers in existing genetic maps has limited the efficiency and accuracy of QTL mapping. Genotyping by sequencing (GBS) is an improved strategy for addressing a complex genome via next-generation sequencing technology. GBS has been a powerful tool for SNP discovery and high-density genetic map construction. The creation of ultra-high density genetic maps using large populations of advanced recombinant inbred lines (RILs) is an efficient way to identify QTL for complex agronomic traits. RESULTS: A set of 314 RILs derived from inbreds Ye478 and Qi319 were generated and subjected to GBS. A total of 137,699,000 reads with an average of 357,376 reads per individual RIL were generated, which is equivalent to approximately 0.07-fold coverage of the maize B73 RefGen_V3 genome for each individual RIL. A high-density genetic map was constructed using 4183 bin markers (100-Kb intervals with no recombination events). The total genetic distance covered by the linkage map was 1545.65 cM and the average distance between adjacent markers was 0.37 cM with a physical distance of about 0.51 Mb. Our results demonstrated a relatively high degree of collinearity between the genetic map and the B73 reference genome. The quality and accuracy of the bin map for QTL detection was verified by the mapping of a known gene, pericarp color 1 (P1), which controls the color of the cob, with a high LOD value of 80.78 on chromosome 1. Using this high-density bin map, 35 QTL affecting plant architecture, including 14 for plant height, 14 for ear height, and seven for internode number were detected across three environments. Interestingly, pQTL10, which influences all three of these traits, was stably detected in three environments on chromosome 10 within an interval of 14.6 Mb. Two MYB transcription factor genes, GRMZM2G325907 and GRMZM2G108892, which might regulate plant cell wall metabolism are the candidate genes for qPH10. CONCLUSIONS: Here, an ultra-high density accurate linkage map for a set of maize RILs was constructed using a GBS strategy. This map will facilitate identification of genes and exploration of QTL for plant architecture in maize. It will also be helpful for further research into the mechanisms that control plant architecture while also providing a basis for marker-assisted selection.