Near-isogenic Line Population Research Articles

GL5.2, a Quantitative Trait Locus for Rice Grain Shape, Encodes a RING-Type E3 Ubiquitin Ligase.

Grain weight and grain shape are important traits that determine rice grain yield and quality. Mining more quantitative trait loci (QTLs) that control grain weight and shape will help to further improve the molecular regulatory network of rice grain development and provide gene resources for high-yield and high-quality rice varieties. In the present study, a QTL for grain length (GL) and grain width (GW), qGL5.2, was firstly fine-mapped into a 21.4 kb region using two sets of near-isogenic lines (NILs) derived from the indica rice cross Teqing (TQ) and IRBB52. In the NIL populations, the GL and ratio of grain length to grain width (RLW) of the IRBB52 homozygous lines increased by 0.16-0.20% and 0.27-0.39% compared with the TQ homozygous lines, but GW decreased by 0.19-0.75%. Then, by analyzing the grain weight and grain shape of the knock-out mutant, it was determined that the annotation gene Os05g0551000 encoded a RING-type E3 ubiquitin ligase, which was the cause gene of qGL5.2. The results show that GL and RLW increased by 2.44-5.48% and 4.19-10.70%, but GW decreased by 1.69-4.70% compared with the recipient. Based on the parental sequence analysis and haplotype analysis, one InDel variation located at -1489 in the promoter region was likely to be the functional site of qGL5.2. In addition, we also found that the Hap 5 (IRBB52-type) increased significantly in grain length and grain weight compared with other haplotypes, indicating that the Hap 5 can potentially be used in rice breeding to improve grain yield and quality.

Fine mapping and transcriptome sequencing reveal candidate genes conferring all-stage resistance to stripe rust on chromosome arm 1AL in Chinese wheat landrace AS1676

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), threatens wheat production worldwide, and resistant varieties tend to become susceptible after a period of cultivation owing to the variation of pathogen races. In this study, a new resistance gene against Pst race CYR34 was identified and predicted using the descendants of a cross between AS1676, a highly resistant Chinese landrace, and Avocet S, a susceptible cultivar. From a heterozygous plant from a F7 recombinant inbred line (RIL) population lacking the Yr18 gene, a near-isogenic line (NIL) population was developed to map the resistance gene. An all-stage resistance gene, YrAS1676, was identified on chromosome arm 1AL via bulked-segregant exome-capture sequencing. By analyzing a large NIL population consisting of 6537 plants, the gene was further mapped to the marker interval between KA1A_485.36 and KA1A_490.13, spanning 485.36–490.13 Mb on 1AL. A total of 66 annotated genes have been reported in this region. To characterize and predict the candidate gene(s), an RNA-seq was performed using NIL-R and NIL-S seedlings 3 days after CYR34 inoculation. Compared to NIL-S plants, NIL-R plants showed stronger immune reaction and higher expression levels of genes encoding pathogenesis-associated proteins. These differences may help to explain why NIL-R plants were more resistant to Pst race CYR34 than NIL-S plants. By combining fine-mapping and transcriptome sequencing, a calcium-dependent protein kinase gene was finally predicted as the potential candidate gene of YrAS1676. This gene contained a single-nucleotide polymorphism. The candidate gene was more highly expressed in NIL-R than in NIL-S plants. In field experiments with Pst challenge, the YrAS1676 genotype showed mitigation of disease damage and yield loss without adverse effects on tested agronomic traits. These results suggest that YrAS1676 has potential use in wheat stripe rust resistance breeding.

Dissection of two quantitative trait loci for grain length linked on the long arm of chromosome 5 in rice

AbstractGrain size is the major determinant of grain weight and has an important role in grain quality. It is controlled by several major quantitative trait loci (QTL) and many minor QTL. Identification of QTL for grain size is important for understanding the genetic and molecular network regulating grain size in rice (Oryza sativa L.). Following previous identification of QTL for grain weight and size using an F2:3 family derived from the indica rice cross Teqing/IRBB52, one QTL, qTGW5, having significant effects on grain length and weight, was targeted for validation, dissection, and fine‐mapping. First, the effect of qTGW5 was validated using two near‐isogenic line (NIL)‐F2 populations. Then, qTGW5 was dissected into two closely linked QTL using four NIL populations. One of them, qGL5.1, having significant effects on grain length, width, and weight, was located within an 1,896.4‐kb region. The other one, qGL5.2, controlling grain length, was further delimited into a 68.8‐kb region using seven NIL‐F2 populations. Six annotated genes were found in the qGL5.2 region, of which five showed nucleotide polymorphisms between the two parental lines. Three of the six annotated genes showed significant expression differences between NILTeqing and NILIRBB52 in young panicles using quantitative real‐time polymerase chain reaction. The results will facilitate cloning the minor QTL and understanding the genetic architecture for grain size in rice.

Assessment of soybean test weight among genotypes, environments, agronomic and seed compositional traits

AbstractThe value of soybean [Glycine max (L.) Merr.] depends on quantity (yield) and quality, such as seed composition and test weight (TW). Test weight is a bulk density measurement for grain quality evaluation, and higher TW grains are preferred for storage, transport, and export. Thus, soybean breeding should focus on improvement of TW. The objectives of this study were to determine genotypic and environmental effects on TW of soybean and explore relationships of TW with yield, maturity, 100 seed weight (SDWT), seed quality (SQ), seed oil concentration (SOC), and seed protein concentration (SPC). Three sets of breeding populations (BPs), two mapping populations (MPs), and five different high vs. low seed protein near‐isogenic lines populations (NILPs) were used. The BPs and MPs had an average range of 3.5 kg hectoliter (hL)–1 in TW among genotypes. The average ranges of TW in the NILPs were equal for both low protein (LP) and high protein (HP) lines (2.7 kg hL–1). Broad‐sense heritability estimates for TW ranged from .62 to .95. The genotypic and G × E effects on TW were significant in all trials. The SDWT, SQ, SOC, and SPC had strong significant relationships with TW in multiple trials, but were inconsistent overall. Test weight of NILPs differing by presence of ‘Danbaekkong’ high protein allele on chromosome 20 had mostly significant positive and negative relationships with SPC and SOC, respectively.

Fine Mapping and Candidate Gene Prediction of the Quantitative Trait Locus qPL8 for Panicle Length in Rice

Rice panicle is the sink organ where assimilation product accumulates, and its morphology determines the rice yield. Panicle length has been suggested as a yield-related trait, but the genetic factor for its control is still limited. In this study, we carried out fine-mapping of <i>qPL8</i>, a QTL identified for panicle length in our previous work. Near isogenic line (NIL) with qPL8 exhibited elongated panicle without obvious effect on other panicle elements. With five key recombinants from NIL population, the locus was finally narrowed down to a 278-kb region, where 44 genes are annotated. By comparing the genomic sequence of two parents, 17 genes were identified with SNPs or InDels variations in the coding region. Expression analysis showed that eight genes were up-regulated in the NIL with <i>qPL8</i>. Considering both the coding variation and expression status, several candidate genes for the locus were identified, and <i>OsMADS37</i> was raised as the most possible candidate. Interestingly, an expression QTL (eQTL) also resides in the locus, leading to a cluster of gene expression variation in the region. This study will facilitate the application of <i>qPL8</i> locus in rice breeding for yield potential.

Identification through fine mapping and verification using CRISPR/Cas9-targeted mutagenesis for a minor QTL controlling grain weight in rice

Key messageA minor QTL for grain weight in rice, qTGW1.2b, was fine-mapped. Its casual gene OsVQ4 was confirmed through CRISPR/Cas9-targeted mutagenesis, exhibiting an effect that was larger than the original QTL effect.The CRISPR/Cas system exhibits a great potential for rice improvement, but the application was severely hindered due to insufficient target genes, especial the lack of validated genes underlying quantitative trait loci having small effects. In this study, a minor QTL for grain weight, qTGW1.2b, was fine-mapped into a 44.0 kb region using seven sets of near isogenic lines (NILs) developed from the indica rice cross (Zhenshan 97)3/Milyang 46, followed by validation of the causal gene using CRISPR/Cas9-targeted mutagenesis. In the NIL populations, 1000-grain weight of the Zhenshan 97 homozygous lines decreased by 0.9–2.0% compared with the Milyang 46 homozygous lines. A gene encoding VQ-motif protein, OsVQ4, was identified as the candidate gene based on parental sequence differences. The effect of OsVQ4 was confirmed by creating CRISPR/Cas9 knockout lines, whose 1000-grain weight decreased by 2.8–9.8% compared with the wild-type transgenic line and the recipient. These results indicate that applying genome editing system could create novel alleles with large phenotypic variation at minor QTLs, which is an effective way to validate causal genes of minor QTLs. Our study establishes a strategy for cloning minor QTLs, which could also be used to identify a large number of potential target genes for the application of CRISPR/Cas system.

Fine mapping of a novel male-sterile mutant showing wrinkled-leaf in sesame by BSA-Seq technology

Genic male sterility (GMS) is a very important approach of heterosis exploitation in sesame (Sesamum indicum L.). Visible marker-trait linked to GMS is necessary since it can facilitate the breeding of new GMS lines and the production of F1 hybrid seeds. We have successfully developed such a GMS line showing wrinkled-leaf (JQA) from a mutant identified in a multiple-cross population in sesame. However, the underlying genetic basis for this JQA line is unclear. To understand the inheritance patterns, near-isogenic line (NIL) together with F2 and BC1 populations segregating for both pollen-fertility and leaf-shape was constructed. Morphology investigation indicated that the JQA line was controlled by a single recessive gene, Sims5. By combining bulk-segregant analysis and next-generation sequencing technology (BSA-Seq) in the NIL population, seven and four chromosome regions putatively associated with traits variation were identified by SNP and InDel markers, respectively. A 1,817.6-kb consensus region on chromosome 12 was obtained by intersecting these intervals, which was most likely the target region containing the candidate gene(s) responsible for pollen-fertility and leaf-shape. The consensus region was further narrowed down to only 219.7 kb by fine mapping in a BC1 population using SSR and InDel markers. Real-Time qRT-PCR analysis of genes within this region showed that four (XM_011097706.2, XM_011097714.2, XM_020698411.1 and XM_011097719.2) and one (XM_020698321.1) genes were highly suppressed in the anther and leaf of mutant plant, respectively, suggesting their important role in pollen and leaf development. These findings not only lay a solid foundation for the isolation and functional analysis of genes for pollen-fertility and wrinkled-leaf but also provided valuable genetic resources and molecular markers for the breeding of new sesame hybrid varieties.

Fine-mapping of qTGW2, a quantitative trait locus for grain weight in rice (Oryza sativa L.).

BackgroundGrain weight is a grain yield component, which is an integrated index of grain length, width and thickness. They are controlled by a large number of quantitative trait loci (QTLs). Besides major QTLs, minor QTLs play an essential role. In our previous studies, QTL analysis for grain length and width was performed using a recombinant inbred line population derived from rice cross TQ/IRBB lines. Two major QTLs were detected, which were located in proximity to GS3 and GW5 that have been cloned. In the present study, QTLs for grain weight and shape were identified using rice populations that were homozygous at GS3 and GW5.MethodNine populations derived from the indica rice cross TQ/IRBB52 were used. An F10:11population named W1, consisting of 250 families and covering 16 segregating regions, was developed from one residual heterozygote (RH) in the F7generation of Teqing/IRBB52. Three near isogenic line (NIL)-F2 populations, ZH1, ZH2 and ZH3 that comprised 205, 239 and 234 plants, respectively, were derived from three RHs in F10:11. They segregated the target QTL region in an isogenic background. Two NIL populations, HY2 and HY3, were respectively produced from homozygous progeny of the ZH2 and ZH3 populations. Three other NIL-F2 populations, Z1, Z2 and Z3, were established using three RHs having smaller heterozygous segments. QTL analysis for 1000-grain weight (TGW), grain length (GL), grain width (GW), and length/width ratio (LWR) was conducted using QTL IciMapping and SAS procedure with GLM model.ResultA total of 27 QTLs distributed on 12 chromosomes were identified. One QTL cluster, qTGW2/qGL2/qGW2 located in the terminal region of chromosome 2, were selected for further analysis. Two linked QTLs were separated in region Tw31911−RM266. qGL2 was located in Tw31911−Tw32437 and mainly controlled GL and GW. The effects were larger on GL than on GW and the allelic directions were opposite. qTGW2 was located in Tw35293−RM266 and affected TGW, GL and GW with the same allelic direction. Finally, qTGW2 was delimited within a 103-kb region flanked by Tw35293 and Tw35395.ConclusionqTGW2 with significant effects on TGW, GL and GW was validated and fine-mapped using NIL and NIL-F2 populations. These results provide a basis for map-based cloning of qTGW2 and utilization of qTGW2 in the breeding of high-yielding rice varieties.

Pleiotropic Effects of Rice Florigen Gene RFT1 on the Amino Acid Content of Unmilled Rice.

In rice, the contents of protein and amino acids are the major parameters of nutritional quality. Co-localization of quantitative trait loci (QTLs) for heading date and protein content were reported, but pleiotropism of heading-date genes on protein contents has not been investigated. Here, we reported that rice florigen gene RFT1 plays an important role in controlling amino acid contents of rice grain. Firstly, 73 QTLs for the contents of 17 amino acids in unmilled rice were detected using recombinant inbred lines (RILs) of the indica rice cross Zhenshan 97 (ZS97)/Milyang 46 (MY46). Then, the effect of the largest cluster consisting of 14 QTLs, located in proximity to the rice florigen genes RFT1 and Hd3a, was validated using three populations consisting of near isogenic lines (NILs) that only segregated a region covering the target QTL. The first and second NIL populations were derived from a residual heterozygote identified from the ZS97/MY46 RIL population, consisting of homozygous lines that were only segregated in a 29.9-kb region covering the two florigen genes and a 1.7-kb region for RFT1, respectively. The third NIL population was segregated for the RFT1 ZS97 transgene in the background of japonica rice cultivar Zhonghua 11. In all the three NIL populations, RFT1 was shown to have a strong effect on the contents of most amino acids, with the ZS97 allele always having the reducing effects. By comparing QTLs for amino acid contents detected in the ZS97/MY46 RIL population and genes/QTLs previously identified for heading date difference between ZS97 and MY46, possible pleiotropism on amino acid contents was also shown for other key heading-date genes including Hd1, Ghd7, and OsGI.

Allelic Variation in the Chloroplast Division Gene FtsZ2-2 Leads to Natural Variation in Chloroplast Size.

Chloroplast size varies considerably in nature, but the underlying mechanisms are unknown. By exploiting a near-isogenic line population derived from a cross between the Arabidopsis (Arabidopsis thaliana) accessions Cape Verde Islands (Cvi-1), which has larger chloroplasts, and Landsberg erecta (Ler-0), with smaller chloroplasts, we determined that the large-chloroplast phenotype in Cvi-1 is associated with allelic variation in the gene encoding the chloroplast-division protein FtsZ2-2, a tubulin-related cytoskeletal component of the contractile FtsZ ring inside chloroplasts. Sequencing revealed that the Cvi-1 FtsZ2-2 allele encodes a C-terminally truncated protein lacking a region required for FtsZ2-2 interaction with inner-envelope proteins, and functional complementation experiments in a Columbia-0 ftsZ2-2 null mutant confirmed this allele as causal for the increased chloroplast size in Cvi-1. Comparison of FtsZ2-2 coding sequences in the 1001 Genomes database showed that the Cvi-1 allele is rare and identified additional rare loss-of-function alleles, including a natural null allele, in three other accessions, all of which had enlarged-chloroplast phenotypes. The ratio of nonsynonymous to synonymous substitutions was higher among the FtsZ2-2 genes than among the two other FtsZ family members in Arabidopsis, FtsZ2-1, a close paralog of FtsZ2-2, and the functionally distinct FtsZ1-1, indicating more relaxed constraint on the FtsZ2-2 coding sequence than on those of FtsZ2-1 or FtsZ1-1 Our results establish that allelic variation in FtsZ2-2 contributes to natural variation in chloroplast size in Arabidopsis, and they also demonstrate that natural variation in Arabidopsis can be used to decipher the genetic basis of differences in fundamental cell biological traits, such as organelle size.

Fine-Mapping of qTGW1.2a, a Quantitative Trait Locus for 1000-Grain Weight in Rice

Thousand-grain weight (TGW) is a key component of grain yield in rice. This study was conducted to validate and fine-map qTGW1.2a, a quantitative trait locus for grain weight and grain size previously located in a 933.6-kb region on the long arm of rice chromosome 1. Firstly, three residual heterozygotes (RHs) were selected from a BC2F11 population of the indica rice cross Zhenshan 97 (ZS97)///ZS97//ZS97/Milyang 46. The heterozygous segments in these RHs were arranged successively in physical positions, forming one set of sequential residual heterozygotes (SeqRHs). In each of the populations derived, non-recombinant homozygotes were identified to produce near isogenic lines (NILs) comprising the two homozygous genotypes. The NILs were tested for grain weight, grain length and grain width. QTL analyses for the three traits were performed. Then, the updated QTL location was followed for a new run of SeqRHs identification-NIL development-QTL mapping. Altogether, 11 NIL populations derived from four sets of SeqRHs were developed and used. qTGW1.2a was finally delimitated into a 77.5-kb region containing 13 annotated genes. In the six populations segregating this QTL, which were in four generations and were tested across four years, the allelic direction of qTGW1.2a remained consistent and the genetic effects were stable. For TGW, the additive effects ranged from 0.23 to 0.38g and the proportions of phenotypic variance explained ranged from 26.15% to 41.65%. These results provide a good foundation for the cloning and functional analysis of qTGW1.2a.

Dissection of three quantitative trait loci for grain size on the long arm of chromosome 10 in rice (Oryza sativa L.).

BackgroundThousand grain weight is a key component of grain yield in rice, and a trait closely related to grain length (GL) and grain width (GW) that are important traits for grain quality. Causal genes for 16 quantitative trait loci (QTL) affecting these traits have been cloned, but more QTL remain to be characterized for establishing a genetic regulating network. A QTL controlling grain size in rice, qGS10, was previously mapped in the interval RM6100–RM228 on chromosome 10. This study aimed to delimitate this QTL to a more precise location.MethodA total of 12 populations were used. The ZC9 population comprised 203 S1:2 families derived from a residual heterozygous (RH) plant in the F9 generation of the indica rice cross Teqing (TQ)/IRBB52, segregating the upper region of RM6100–RM228 and three more regions on chromosomes 1, 9, and 11. The Ti52-1 population comprised 171 S1 plants derived from one RH plant in F7 of TQ/IRBB52, segregating a single interval that was in the lower portion of RM6100–RM228. The other ten populations were all derived from Ti52-1, including five S1 populations with sequential segregating regions covering the target region and five near isogenic line (NIL) populations maintaining the same segregating pattern. QTL analysis for 1,000-grain weight, GL, and GW was performed using QTL IciMapping and SAS procedure GLM.ResultThree QTL were separated in the original qGS10 region. The qGL10.1 was located in the upper region RM6704–RM3773, shown to affect GL only. The qGS10.1 was located within a 207.1-kb interval flanked by InDel markers Te20811 and Te21018, having a stable and relatively high effect on all the three traits analyzed. The qGS10.2 was located within a 1.2-Mb interval flanked by simple sequence repeat markers RM3123 and RM6673. This QTL also affected all the three traits but the effect was inconsistent across different experiments. QTL for grain size were also detected in all the other three segregating regions.ConclusionThree QTL for grain size that were tightly linked on the long arm of chromosome 10 of rice were separated using NIL populations with sequential segregating regions. One of them, qGS10.1, had a stable and relatively high effect on grain weight, GL, and GW, providing a good candidate for gene cloning. Another QTL, qGS10.2, had a significant effect on all the three traits but the effect was inconsistent across different experiments, providing an example of genotype-by-environmental interaction.

Fine mapping of the major QTL for seed coat color in Brassica rapa var. Yellow Sarson by use of NIL populations and transcriptome sequencing for identification of the candidate genes.

Yellow seed is a desirable trait in Brassica oilseed crops. The B. rapa var. Yellow Sarson carry unique yellow seed color genes which are not only important for the development of yellow-seeded oilseed B. rapa cultivars but this variant can also be used to develop yellow-seeded B. napus. In this study, we developed near-isogenic lines (NILs) of Yellow Sarson for the major seed coat color QTL SCA9-2 of the chromosome A9 and used the NILs to fine map this QTL region and to identify the candidate genes through linkage mapping and transcriptome sequencing of the developing seeds. From the 18.4 to 22.79 Mb region of SCA9-2, six SSR markers showing 0.63 to 5.65% recombination were developed through linkage analysis and physical mapping. A total of 55 differentially expressed genes (DEGs) were identified in the SCA9-2 region through transcriptome analysis; these included three transcription factors, Bra028039 (NAC), Bra023223 (C2H2 type zinc finger), Bra032362 (TIFY), and several other genes which encode unknown or nucleic acid binding protein; these genes might be the candidates and involved in the regulation of seed coat color in the materials used in this study. Several biosynthetic pathways, including the flavonoid, phenylpropanoid and suberin biosynthetic pathways were significantly enriched through GO and KEGG enrichment analysis of the DEGs. This is the first comprehensive study to understand the yellow seed trait of Yellow Sarson through employing linkage mapping and global transcriptome analysis approaches.

Genetic analysis of cob resistance to F. verticillioides: another step towards the protection of maize from ear rot.

We lay the foundation for further research on maize resistance to Fusarium verticillioides cob rot by identifying a candidate resistance gene. Fusarium verticillioides ear rot is the most common type of maize ear rot in the Huanghuaihai Plain of China. Ear rot resistance includes cob and kernel resistance. Most of the current literature concentrates on kernel resistance, and genetic studies on cob resistance are scarce. We aimed on identifying the QTLs responsible for F. verticillioides cob rot (FCR) resistance. Twenty-eight genes associated with 48 single nucleotide polymorphisms (SNPs) were identified (P < 10-4) to correlate with FCR resistance using a whole-genome association study. The major quantitative trait locus, qRcfv2, for FCR resistance was identified on chromosome 2 through linkage mapping and was validated in near-isogenic line populations. Two candidate genes associated with two SNPs were detected in the qRcfv2 region with a lower threshold (P < 10-3). Through real-time fluorescence quantitative PCR, one candidate gene was found to have no expression in the cob but the other was expressed in response to F. verticillioides. These results lay a foundation for research on the resistance mechanisms of cob and provide resources for marker-assisted selection.

Fine mapping quantitative trait loci that underlie resistance to soybean sudden death syndrome using NILs and SNPs.

Soybean (Glycine max (L.) Merr.) cultivars differ in their resistance to sudden death syndrome (SDS), caused by Fusarium virguliforme. Breeding for improving SDS response has been challenging, due to interactions among the 18-42 known resistance loci. Four quantitative trait loci (QTL) for resistance to SDS (cqRfs–cqRfs3) were clustered within 20 cM of the rhg1 locus underlying resistance to soybean cyst nematode (SCN) on Chromosome (Chr.) 18. Another locus on Chr. 20 (cqRfs5) was reported to interact with this cluster. The aims here were to compare the inheritance of resistance to SDS in a near isogenic line (NIL) population that was fixed for resistance to SCN but segregated at two of the four loci (cqRfs1 and cqRfs) for SDS resistance; to examine the interaction with the locus on Chr. 20; and to identify candidate genes underlying QTL. Used were; a NIL population derived from residual heterozygosity in an F5:7 recombinant inbred line EF60 (lines 1-38); SDS response data from two locations and years; four segregating microsatellite and 1,500 SNP markers. Polymorphic regions were found from 2,788 Kbp to 8,938 Kbp on Chr. 18 and 33,100 Kbp to 34,943 Kbp on Chr. 20 that were significantly (0.005 &lt; P &gt; 0.0001) associated with resistance to SDS. The QTL fine maps suggested that the two loci on Chr. 18 were three loci (cqRfs1, cqRfs, and cqRfs19). Candidate genes were inferred. An epistatic interaction was inferred between Chr. 18 and Chr. 20 loci. Therefore, SDS resistance QTL were both complex and interacting.

The effect of stem growth habit on single seed weight and seed uniformity in soybean (Glycine max (L.) Merrill).

The timing of flower formation and length of the seed-filling period of indeterminate growth soybean varieties vary more than those of determinate varieties (Glycine max (L.) Merrill). These variations have been hypothesized to affect single seed weight and its uniformity which determine the processing quality of soybean used in foods. We derived near isogenic lines (NILs) with different growth characteristics from an indeterminate line (donor parent) and three determinate lines with heavy seeds (recurrent parents), and evaluated the effects of growth habit on seed weight and its uniformity. Each NIL population consisting of five indeterminate and five determinate BC4F4 lines tested at two locations in two different years with two replications. Split-plot analysis of variance, with main-plot and sub-plot being cross combination and growth habit, respectively, showed that indeterminate varieties had slightly heavier seeds than determinate varieties and that there was no significant difference in uniformity of single seed weights. The effects of growth habit on seed uniformity was related to genetic background, but differences between the two growth characteristics were less than the differences among genetic background. This indicates that indeterminate growth habit did not much influence seed weight or its uniformity.

Identification of Teosinte Alleles for Resistance to Southern Leaf Blight in Near Isogenic Maize Lines

Southern leaf blight ([SLB], causal agent Cochliobolus heterostrophus) is an important fungal disease of maize (Zea mays L.). Teosinte (Z. mays ssp. parviglumis), the wild progenitor of maize, offers a novel source of resistance alleles that may have been lost during domestication. The aims of this study were to identify teosinte alleles that, when present in a temperate maize background, confer a significant level of resistance to SLB. Ten populations of BC4S2 near isogenic lines (NILs), developed by crossing 10 different teosinte accessions to the maize inbred B73, comprising 774 lines in total, were screened for SLB resistance. Quantitative trait locus (QTL) analysis identified four significant QTL associated with SLB in bins 2.04, 3.04, 3.05, and 8.05. Sixteen individual NILs which were significantly different to the susceptible recurrent parent, B73 and which were carrying at least one of the teosinte‐derived resistance alleles were used to develop F2:3 populations by crossing each to B73 followed by two rounds of self‐pollination. These F2:3 populations were evaluated for SLB resistance and genotyped at the loci of interest. In 13 of 19 cases single marker analysis validated allelic substitution effects predicted from the original NIL population analysis, while in five cases we were not able to validate the effects and in one case a significant effect was detected in the opposite to the predicted direction. An allele at the QTL in bin 2.04 was shown to confer resistance to both SLB and a second maize foliar disease, gray leaf spot (GLS).

Identification of Alleles Conferring Resistance to Gray Leaf Spot in Maize Derived from its Wild Progenitor Species Teosinte

ABSTRACTGray leaf spot (GLS; causal agent Cercospora zeae‐maydis and Cercospora zeina) is an important maize (Zea mays L.) disease in the United States. Current control methods for GLS include using resistant cultivars, crop rotation, chemical applications, and conventional tillage to reduce inoculum levels. Teosinte (Z. mays subsp. parviglumis) is the wild progenitor of maize and easily forms hybrids with current maize inbreds. The aims of this study were to identify alleles from teosinte that, when introduced into temperate maize germplasm, conferred significant levels of GLS resistance. A population of 693 BC4S2 near isogenic lines (NILs), developed by crossing nine different teosinte accessions into the background of the maize inbred B73, were evaluated for GLS resistance in replicated field trials over 2 yr. Six markers significantly associated with GLS resistance were identified using 768 single nucleotide polymorphism (SNP) markers used to genotype this population. Twenty‐seven individual NILs that differed significantly from B73 for GLS resistance and that carried teosinte introgressions at the significantly associated SNPs at bins 2.04, 3.06, 4.07, 5.03, 8.06, and 9.03 were selected for follow‐up studies. F2:3 populations were developed by crossing each selected NIL to B73 followed by self‐pollinating the progeny twice. These F2:3 populations were evaluated for GLS resistance and genotyped at the loci of interest. In most cases, single‐marker analysis validated predicted allelic substitution effects from the original NIL populations.

Mapping of qTGW1.1, a Quantitative Trait Locus for 1000-Grain Weight in Rice (Oryza sativa L.)

1000-grain weight (TGW) is one of the three component traits of the grain yield in rice (Oryza sativa L). This study was conducted to validate and fine-map qTGW1.1, a minor QTL for TGW which was previously located in a 3.7-Mb region on the long arm of rice chromosome 1. Five sets of near isogenic lines (NILs) were developed from two BC2F4 populations of the indica rice cross Zhenshan 973/Milyang 46. The NIL sets consisted of two homozygous genotypic groups differing in the regions RM11448−RM11522, RM11448−RM11549, RM1232−RM11615, RM11543−RM11554 and RM11569−RM11621, respectively. Four traits, including TGW, grain length, grain width and heading date, were measured. Phenotypic difference between the two genotypic groups in each NIL population was analyzed using SAS procedure GLM. Significant QTL effects were detected on TGW with the Zhenshan 97 allele increasing grain weight by 0.12g to 0.14g and explaining 8.30% to 15.19% of the phenotypic variance. Significant effects were also observed for grain length and width, whereas no significant effect was found for heading date. Based on comparison among the five NILs on the segregating regions and the results of QTL analysis, qTGW1.1 was delimited to a 376.9-kb region flanked by DNA markers Wn28382 and RM11554. Our results indicate that the effects of minor QTLs could be steadily detected in a highly isogenic background and suggest that such QTLs could be utilized in the breeding of high-yielding rice varieties.

Inheritance Patterns and Stability of DNA Methylation Variation in Maize Near-Isogenic Lines

DNA methylation is a chromatin modification that contributes to epigenetic regulation of gene expression. The inheritance patterns and trans-generational stability of 962 differentially methylated regions (DMRs) were assessed in a panel of 71 near-isogenic lines (NILs) derived from maize (Zea mays) inbred lines B73 and Mo17. The majority of DMRs exhibit inheritance patterns that would be expected for local (cis) inheritance of DNA methylation variation such that DNA methylation level was coupled to local genotype. There are few examples of DNA methylation that exhibit trans-acting control or paramutation-like patterns. The cis-inherited DMRs provide an opportunity to study the stability of inheritance for DNA methylation variation. There was very little evidence for alterations of DNA methylation levels at these DMRs during the generations of the NIL population development. DNA methylation level was associated with local genotypes in nearly all of the >30,000 potential cases of inheritance. The majority of the DMRs were not associated with small RNAs. Together, our results suggest that a significant portion of DNA methylation variation in maize exhibits locally (cis) inherited patterns, is highly stable, and does not require active programming by small RNAs for maintenance. DNA methylation may contribute to heritable epigenetic information in many eukaryotic genomes. In this study, we have documented the inheritance patterns and trans-generational stability for nearly 1000 DNA methylation variants in a segregating maize population. At most loci studied, the DNA methylation differences are locally inherited and are not influenced by the other allele or other genomic regions. The inheritance of DNA methylation levels across generations is quite robust with almost no examples of unstable inheritance, suggesting that DNA methylation differences can be quite stably inherited, even in segregating populations.