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.

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.

ZmADF5, a Maize Actin-Depolymerizing Factor Conferring Enhanced Drought Tolerance in Maize.

Drought stress is seriously affecting the growth and production of crops, especially when agricultural irrigation still remains quantitatively restricted in some arid and semi-arid areas. The identification of drought-tolerant genes is important for improving the adaptability of maize under stress. Here, we found that a new member of the actin-depolymerizing factor (ADF) family; the ZmADF5 gene was tightly linked with a consensus drought-tolerant quantitative trait locus, and the significantly associated signals were detected through genome wide association analysis. ZmADF5 expression could be induced by osmotic stress and the application of exogenous abscisic acid. Its overexpression in Arabidopsis and maize helped plants to keep a higher survival rate after water-deficit stress, which reduced the stomatal aperture and the water-loss rate, as well as improved clearance of reactive oxygen species. Moreover, seventeen differentially expressed genes were identified as regulated by both drought stress and ZmADF5, four of which were involved in the ABA-dependent drought stress response. ZmADF5-overexpressing plants were also identified as sensitive to ABA during the seed germination and seedling stages. These results suggested that ZmADF5 played an important role in the response to drought stress.

A transcription factor ZmGLK36 confers broad resistance to maize rough dwarf disease in cereal crops.

Maize rough dwarf disease (MRDD), caused by maize rough dwarf virus (MRDV) or rice black-streaked dwarf virus (RBSDV), seriously threatens worldwide production of all major cereal crops, including maize, rice, wheat and barley. Here we report fine mapping and cloning of a previously reported major quantitative trait locus (QTL) (qMrdd2) for RBSDV resistance in maize. Subsequently, we show that qMrdd2 encodes a G2-like transcription factor named ZmGLK36 that promotes resistance to RBSDV by enhancing jasmonic acid (JA) biosynthesis and JA-mediated defence response. We identify a 26-bp indel located in the 5' UTR of ZmGLK36 that contributes to differential expression and resistance to RBSDV in maize inbred lines. Moreover, we show that ZmDBF2, an AP2/EREBP family transcription factor, directly binds to the 26-bp indel and represses ZmGLK36 expression. We further demonstrate that ZmGLK36 plays a conserved role in conferring resistance to RBSDV in rice and wheat using transgenic or marker-assisted breeding approaches. Our results provide insights into the molecular mechanisms of RBSDV resistance and effective strategies to breed RBSDV-resistant cereal crops.

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.

ZmSNAC13, a maize NAC transcription factor conferring enhanced resistance to multiple abiotic stresses in transgenic Arabidopsis.

Abiotic stress is the main factor that severely limits crop growth and yield. NAC (NAM, ATAF1/2 and CUC2) transcription factors play an important role in dealing with various abiotic stresses. Here, we discovered the ZmSNAC13 gene in drought-tolerant maize lines by RNA-seq analysis and verified its function in Arabidopsis thaliana. First, its gene structure showed that ZmSNAC13 had a typical NAC domain and a highly variable C-terminal. There were multiple cis-acting elements related to stress in its promoter region. Overexpression of ZmSNAC13 resulted in enhanced tolerances to drought and salt stresses in Arabidopsis, characterized by a reduction in the water loss rate, a sustained effective photosynthesis rate, and increased cell membrane stability in leaves under drought conditions. Transcriptome analysis showed that a large number of differentially expressed genes regulated by overexpression of ZmSNAC13 were identified, and the main drought tolerance regulatory pathways involved were the ABA pathway and MAPK cascade signaling pathway. Overexpression of ZmSNAC13 promoted the expression of genes, such as PYL9 and DREB3, thereby enhancing tolerance to adverse environments. Adaptability, while restraining genes expression such as WRKY53 and MPK3, facilitates regulation of senescence in Arabidopsis and improves plant responses to adversity. Therefore, ZmSNAC13 is promising gene of interest for use in transgenic breeding to improve abiotic stress tolerance in crops.

Genetic basis of maize kernel protein content revealed by high-density bin mapping using recombinant inbred lines.

Maize with a high kernel protein content (PC) is desirable for human food and livestock fodder. However, improvements in its PC have been hampered by a lack of desirable molecular markers. To identify quantitative trait loci (QTL) and candidate genes for kernel PC, we employed a genotyping-by-sequencing strategy to construct a high-resolution linkage map with 6,433 bin markers for 275 recombinant inbred lines (RILs) derived from a high-PC female Ji846 and low-PC male Ye3189. The total genetic distance covered by the linkage map was 2180.93 cM, and the average distance between adjacent markers was 0.32 cM, with a physical distance of approximately 0.37 Mb. Using this linkage map, 11 QTLs affecting kernel PC were identified, including qPC7 and qPC2-2, which were identified in at least two environments. For the qPC2-2 locus, a marker named IndelPC2-2 was developed with closely linked polymorphisms in both parents, and when tested in 30 high and 30 low PC inbred lines, it showed significant differences (P = 1.9E-03). To identify the candidate genes for this locus, transcriptome sequencing data and PC best linear unbiased estimates (BLUE) for 348 inbred lines were combined, and the expression levels of the four genes were correlated with PC. Among the four genes, Zm00001d002625, which encodes an S-adenosyl-L-methionine-dependent methyltransferase superfamily protein, showed significantly different expression levels between two RIL parents in the endosperm and is speculated to be a potential candidate gene for qPC2-2. This study will contribute to further research on the mechanisms underlying the regulation of maize PC, while also providing a genetic basis for marker-assisted selection in the future.

Transcriptome Reveals Allele Contribution to Heterosis in Maize.

Heterosis, which has greatly increased maize yields, is associated with gene expression patterns during key developmental stages that enhance hybrid phenotypes relative to parental phenotypes. Before heterosis can be more effectively used for crop improvement, hybrid maize developmental gene expression patterns must be better understood. Here, six maize hybrids, including the popular hybrid Zhengdan958 (ZC) from China, were studied. Maize hybrids created in-house were generated using an incomplete diallel cross (NCII)-based strategy from four elite inbred parental lines. Differential gene expression (DEG) profiles corresponding to three developmental stages revealed that hybrid partial expression patterns exhibited complementarity of expression of certain parental genes, with parental allelic expression patterns varying both qualitatively and quantitatively in hybrids. Single-parent expression (SPE) and parent-specific expression (PSE) types of qualitative variation were most prevalent, 43.73 and 41.07% of variation, respectively. Meanwhile, negative super-dominance (NSD) and positive super-dominance (PSD) types of quantitative variation were most prevalent, 31.06 and 24.30% of variation, respectively. During the early reproductive growth stage, the gene expression pattern differed markedly from other developmental stage patterns, with allelic expression patterns during seed development skewed toward low-value parental alleles in hybrid seeds exhibiting significant quantitative variation-associated superiority. Comparisons of qualitative gene expression variation rates between ZC and other hybrids revealed proportions of SPE-DEGs (41.36%) in ZC seed DEGs that significantly exceeded the average proportion of SPE-DEGs found in seeds of other hybrids (28.36%). Importantly, quantitative gene expression variation rate comparisons between ZC and hybrids, except for transgressive expression, revealed that the ZC rate exceeded the average rate for other hybrids, highlighting the importance of partial gene expression in heterosis. Moreover, enriched ZC DEGs exhibiting distinct tissue-specific expression patterns belonged to four biological pathways, including photosynthesis, plant hormone signal transduction, biology metabolism and biosynthesis. These results provide valuable technical insights for creating hybrids exhibiting strong heterosis.

Natural variations in the non-coding region of ZmNAC080308 contributes maintaining grain yield under drought stress in maize.

BACKGROUND: Natural variations derived from both evolutionary selection and genetic recombination, presume to have important functions to respond to various abiotic stresses, which could be used to improve drought tolerance via genomic selection. RESULTS: In the present study, the NAC-encoding gene of ZmNAC080308 was cloned and sequenced in 199 inbred lines in maize. Phylogenetic analysis showed that ZmNAC080308 is closely clusteredinto the same group with other well-known NAC genes responding to improve drought tolerance. In total, 86 SNPs and 47 InDels were identified in the generic region of ZmNAC080308, 19 of these variations were associated with GY (grain yield) in different environments. Nine variations in the 5'-UTR region of ZmNAC080308 are closely linked, they might regulate the gene expression and respond to improve GY under drought condition via Sp1-mediated transactivation. Two haplotypes (Hap1 and Hap2) identified in the, 5'-UTR region using the nine variations, and Hap2 containing insertion variants, exhibited 15.47 % higher GY under drought stress condition. Further, a functional marker was developed to predict the drought stress tolerance in a US maize inbred line panel. Lines carrying Hap2 exhibited > 10 % higher GY than those carrying Hap1 under drought stress condition. In Arabidopsis, overexpression ZmNAC080308 enhanced drought tolerance. CONCLUSIONS: ZmNAC080308 is an important gene responding to drought tolerance, a functional marker is developed for improving maize drought tolerance by selecting this gene.

Mapping quantitative trait loci associated with stem-related traits in maize (Zea mays L.).

KEY MESSAGE: Mapping QTL for stem-related traits using RIL population with ultra-high density bin map can better dissect pleiotropic QTL controlling stem architecture that can provide valuable information for maize genetic improvement. The maize stem is one of the most important parts of the plant and is also a component of many agronomic traits in maize. This study aimed to advance our understanding of the genetic mechanisms underlying maize stem traits. A recombinant inbred line (RIL) population derived from a cross between Ye478 and Qi319 was used to identify quantitative trait loci (QTL) controlling stem height (SH), ear height (EH), stem node number (SN), ear node (EN), and stem diameter (SD), and two derived traits (ear height coefficient (EHc) and ear node coefficient (ENc)). Using an available ultra-high density bin map, 46 putative QTL for these traits were detected on chromosomes 1, 3, 4, 5, 6, 7, 8, and 10. Individual QTL explained 3.5-17.7% of the phenotypic variance in different environments. Two QTL for SH, three for EH, two for EHc, one for SN, one for EN, and one for SD were detected in more than one environment. QTL with pleiotropic effects or multiple linked QTL were also identified on chromosomes 1, 3, 4, 6, 8, and 10, which are potential target regions for fine-mapping and marker-assisted selection in maize breeding programs. Further, we discussed segregation of bin markers (mk1630 and mk4452) associated with EHc QTL in the RIL population. We had identified two putative WRKY DNA-binding domain proteins, AC209050.3_FG003 and GRMZM5G851490, and a putative auxin response factor, GRMZM2G437460, which might be involved in regulating plant growth and development, as candidate genes for the control of stem architecture.

Genetic Dissection of the General Combining Ability of Yield-Related Traits in Maize.

Maize yield components including row number, kernel number per row, kernel thickness, kernel width, kernel length, 100-kernel weight, and volume weight affect grain yield directly. Previous studies mainly focused on dissecting the genetic basis of per se performances for yield-related traits, but the genetic basis of general combining ability (GCA) for these traits is still unclear. In the present study, 328 RILs were crossed as males to two testers according to the NCII mating design, resulting in a hybrid panel composed of 656 hybrids. Both the hybrids and parental lines were evaluated in four environments in 2015 and 2016. Correlation analysis showed the performances of GCA effects were significantly correlated to the per se performances of RILs for all yield-related traits (0.17 ≤ r ≤ 0.64, P > 0.01). Only 17 of 95 QTL could be detected for both per se performances of RILs and GCA effects for eight yield-related traits. The QTL qKN7-1 and qHKW1-3, which could explain more than 10% of the variation in the GCA effects of KN and HKW, were also detected for per se performances for the traits. The pleiotropic loci qRN3-1 and qRN6, which together explained 14.92% of the observed variation in GCA effects for RN, were associated with the GCA effects of KW and HKW, but not with per se performances for these traits. In contrast, Incw1, which was related to seed weight in maize, was mapped to the region surrounding MK2567 at the qHKW5-2 locus, but no GCA effect was detected. The QTL identified in present study for per se performances and corresponding GCA effects for yield-related traits might be useful for maize hybrid breeding.

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.

Dissecting the Genetic Basis Underlying Combining Ability of Plant Height Related Traits in Maize.

Maize plant height related traits including plant height, ear height, and internode number are tightly linked with biomass, planting density, and grain yield in the field. Previous studies have focused on understanding the genetic basis of plant architecture traits per se, but the genetic basis of combining ability remains poorly understood. In this study, 328 recombinant inbred lines were inter-group crossed with two testers to produce 656 hybrids using the North Carolina II mating design. Both of the parental lines and hybrids were evaluated in two summer maize-growing regions of China in 2015 and 2016. QTL mapping highlighted that 7 out of 16 QTL detected for RILs per se could be simultaneously detected for general combining ability (GCA) effects, suggesting that GCA effects and the traits were genetically controlled by different sets of loci. Among the 35 QTL identified for hybrid performance, 57.1% and 28.5% QTL overlapped with additive/GCA and non-additive/SCA effects, suggesting that the small percentage of hybrid variance due to SCA effects in our design. Two QTL hotspots, located on chromosomes 5 and 10 and including the qPH5-1 and qPH10 loci, were validated for plant height related traits by Ye478 derivatives. Notably, the qPH5-1 locus could simultaneously affect the RILs per se and GCA effects while the qPH10, a major QTL (PVE > 10%) with pleiotropic effects, only affected the GCA effects. These results provide evidence that more attention should be focused on loci that influence combining ability directly in maize hybrid breeding.

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.

Molecular variation and expansion of a rice black-streaked dwarf virus population based on analysis of segment 1 in Jining, China.

To analyze the variation in rice black-streaked dwarf virus (RBSDV) in an area with high incidence of maize rough dwarf disease (MRDD), the RBSDV S1 segment in a collection of 100 maize isolates (sample population A100) from Jining, Shandong Province, was sequenced. An additional 21 maize and rice isolates (subpopulation B21) that were sampled from nine other geographic locations in China in 2012 and 2013 were used as a control. A total of 914 nucleotide mutations, including 239 singleton variable and 675 parsimony-informative sites were detected among the segment 1 (S1) sequences from A100. A total of 614 nucleotide mutation sites including 164 singleton variable and 450 parsimony-informative sites were detected among the S1 sequences from B21, while 97.55 % of the parsimony-informative sites from B21 were also detected in A100. The nucleotide sequence diversities of A100 (π = 0.0479) and B21 (π = 0.0396) were significantly different (P = 0.0002) but showed similar trends. Phylogenetic analysis showed that the 121 RBSDV isolates could be classified into two groups based on their S1 sequences, independent of subpopulation, with a combination of host species and locations. A100 and B21 were under the same level of negative and purifying selection, with Ka/Ks ratios of 0.0337 and 0.0369, respectively. The combined RBSDV population, including 121 isolates, was expanding, with negative values for Tajima's D, Fu and Li's D, and Fu and Li's F in both A100 and B21, except Tajima's D in A100. Based on S1, the RBSDV population in China has long-term phytogeographic stability, and there do not appear to be any newly-emerging strains.

Dual transcriptome analysis reveals insights into the response to Rice black-streaked dwarf virus in maize.

Maize rough dwarf disease (MRDD) is a viral infection that results in heavy yield losses in maize worldwide, particularly in the summer maize-growing regions of China. MRDD is caused by the Rice black-streaked dwarf virus (RBSDV). In the present study, analyses of microRNAs (miRNAs), the degradome, and transcriptome sequences were used to elucidate the RBSDV-responsive pathway(s) in maize. Genomic analysis indicated that the expression of three non-conserved and 28 conserved miRNAs, representing 17 known miRNA families and 14 novel miRNAs, were significantly altered in response to RBSDV when maize was inoculated at the V3 (third leaf) stage. A total of 99 target transcripts from 48 genes of 10 known miRNAs were found to be responsive to RBSDV infection. The annotations of these target genes include a SQUAMOSA promoter binding (SPB) protein, a P450 reductase, an oxidoreductase, and a ubiquitin-related gene, among others. Characterization of the entire transcriptome suggested that a total of 28 and 1085 differentially expressed genes (DEGs) were detected at 1.5 and 3.0 d, respectively, after artificial inoculation with RBSDV. The expression patterns of cell wall- and chloroplast-related genes, and disease resistance- and stress-related genes changed significantly in response to RBSDV infection. The negatively regulated genes GRMZM2G069316 and GRMZM2G031169, which are the target genes for miR169i-p5 and miR8155, were identified as a nucleolin and a NAD(P)-binding Rossmann-fold superfamily protein in maize, respectively. The gene ontology term GO:0003824, including GRMZM2G031169 and other 51 DEGs, was designated as responsive to RBSDV.

Correction: Introgression of opaque2 into Waxy Maize Causes Extensive Biochemical and Proteomic Changes in Endosperm.

[This corrects the article DOI: 10.1371/journal.pone.0158971.].

Fine mapping of a quantitative trait locus conferring resistance to maize rough dwarf disease.

KEY MESSAGE: A QTL qMrdd8 that confers resistance to MRDD was fine mapped into an interval of 347 kb; one SNP and two InDels identified in the interval were significantly associated with resistance to MRDD. Maize rough dwarf disease (MRDD) is highly prevalent in the summer maize-growing areas in China, and leads to significant yield losses in maize (Zea mays L.). In this study, the quantitative trait locus (QTL) qMrdd8, which confers resistance to MRDD, was fine mapped. Initially, qMrdd8 was consistently identified in the interval between the simple sequence repeat markers umc1617 and phi121 in three F2 sub-populations derived from a cross between the resistant recombinant inbred line NL203 and the susceptible line B73. Subsequently, qMrdd8 was fine mapped into an interval of 347 kb defined by the markers IDRQ2 and IDRQ20 using a recombinant-derived progeny test strategy. Based on single nucleotide polymorphism (SNP) genotypes identified using the MaizeSNP50 BeadChip, a long haplotype including qMrdd8 was identified in four resistant inbred lines. One SNP, the 2549-bp insertion/deletion polymorphism (InDel) InDel25, and the 2761-bp InDel27, which all were significantly associated with resistance to MRDD in a set of 226 maize inbred lines (P < 0.05), were detected within qMrdd8. Furthermore, two candidate genes, CG1 and CG2, were detected in the interval using RNA sequencing (RNA-Seq), and InDel25 was localized within the candidate gene CG1. In conclusion, the fine mapping of qMrdd8 will be helpful in cloning the resistance gene, and the three polymorphic markers identified in this study could be used to improve MRDD resistance via a marker-assisted selection approach.

Introgression of opaque2 into Waxy Maize Causes Extensive Biochemical and Proteomic Changes in Endosperm.

Waxy maize is prevalently grown in China and other countries due to the excellent characters and economic value. However, its low content of lysine can't meet the nutritional requirements of humans and livestock. In the present study, we introgressed the opaque2 (o2) allele into waxy maize line Zhao OP-6/O2O2 by using marker-assisted selection (MAS) technique and successfully improved the lysine content and quality of waxy maize. Transcript abundance analysis indicated that the wx1 expression levels had no difference between Zhao OP-6/o2o2 and Zhao OP-6/O2O2. However, Zhao OP-6/o2o2 was characterized by a phenotype of hard and vitreous kernels and accumulation of protein bodies at smaller size (one third of that of parents) but in larger numbers. Biochemical analyses showed that Zhao OP-6/o2o2 had 16.7% less free amino acids than Zhao OP-6/O2O2, especially those derived from glycolytic intermediates, but its content of lysine was increased by 51.6% (0.47% vs. 0.31%). The content of amylopectin was 98.5% in Zhao OP-6/o2o2, significantly higher than that in Zhao OP-6/O2O2 (97.7%). Proteomic analyses indicated that o2 introgression not only decreased the accumulation of various zein proteins except for 27-kDa γ-zein, but also affected other endosperm proteins related to amino acid biosynthesis, starch-protein balance, stress response and signal transduction. This study gives us an intriguing insight into the metabolism changes in endosperm of waxy maize introgressed with opaque2.