Transposon Insertion Drove the Loss of Natural Seed Shattering during Foxtail Millet Domestication.

Loss of seed shattering was a key step during cereal domestication, and it greatly facilitated seed harvest of the staple cereal foxtail millet (Setaria italica) because the cereal has very small seeds. However, the genetic basis for this loss has been largely unknown. Here, we combined comparative and association mapping to identify an 855-bp Harbinger transposable element insertion in the second exon of the foxtail millet gene shattering1 (sh1) that was responsible for the loss of seed shattering. The sh1 gene encodes zinc finger and YABBY domains. The insert prevents transcription of the second exon, causing partial loss of the zinc finger domain and then loss of natural seed shattering. Specifically, sh1 functions as a transcription repressor and represses the transcription of genes associated with lignin synthesis in the abscission zone, including CAD2. The diversity of sh1 is highly reduced in foxtail millet, consistent with either a severe domestication bottleneck or a selective sweep. Phylogenetic analysis of sh1 further revealed a single origin of foxtail millet in China. Our results support the theories that transposons were the most active factors in genome evolution driving loss of natural seed shattering during foxtail millet domestication and that sh1 underwent parallel selection during domestication across different cereal species.

A Large Transposon Insertion in the stiff1 Promoter Increases Stalk Strength in Maize.

Stalk lodging, which is generally determined by stalk strength, results in considerable yield loss and has become a primary threat to maize (Zea mays) yield under high-density planting. However, the molecular genetic basis of maize stalk strength remains unclear, and improvement methods remain inefficient. Here, we combined map-based cloning and association mapping and identified the gene stiff1 underlying a major quantitative trait locus for stalk strength in maize. A 27.2-kb transposable element insertion was present in the promoter of the stiff1 gene, which encodes an F-box domain protein. This transposable element insertion repressed the transcription of stiff1, leading to the increased cellulose and lignin contents in the cell wall and consequently greater stalk strength. Furthermore, a precisely edited allele of stiff1 generated through the CRISPR/Cas9 system resulted in plants with a stronger stalk than the unedited control. Nucleotide diversity analysis revealed that the promoter of stiff1 was under strong selection in the maize stiff-stalk group. Our cloning of stiff1 reveals a case in which a transposable element played an important role in maize improvement. The identification of stiff1 and our edited stiff1 allele pave the way for efficient improvement of maize stalk strength.

A gene regulatory network for tiller development mediated by Tin8 in maize.

The complex gene regulatory network underlying tiller development in maize remains largely unknown. Here we identified two major quantitative trait loci for tiller number, Tin8 on chromosome 8 and the previously known Tb1 on chromosome 1, in a population derived from a teosinte-maize cross. Map-based cloning and association mapping revealed that Tin8, corresponding to Zcn8 encoding a phosphatidylethanolamine-binding-related kinase, is down-regulated in transcription, which results in decreased tiller number. A strong interaction between Tin8 and the key gen Tb1 was detected for tiller number. Further RNA-seq analysis showed that the expression of 13 genes related to tiller development was controlled by Tin8. Our results support the existence of a complex gene regulatory network for the outgrowth of the tiller bud in maize, in which Zcn8 controls 13 tiller-related genes, including four genes for hormonal responses. In particular, Zcn8 represses Gt1, D14, and Tru1 through the interaction with Tb1.

Opposite response of maize ZmCCT to photoperiod due to transposon jumping.

KEY MESSAGE: The new 4.2-kb transposable insertion in the intron of ZmCCT reversely responded relative to the known 5.1-kb transposable insertion to photoperiods between low- and high-latitude regions. Flowering time is a key trait for cereal adaptation that is controlled by a complex genetic background in maize. The effect of multiple alleles from a quantitative trait locus (QTL) on flowering time remains largely unknown. Here, we fine-mapped a major QTL for flowering time on maize chromosome 10 corresponding to ZmCCT, where a new allele with a 4.2-kilobase (kb) transposable insertion was present in the intron. The known allele with a 5.1-kb transposon insertion in the promoter of ZmCCT enhances flowering in high-latitude regions, but has no effect on flowering time in low-latitude regions in comparison with the null allele lacking this insertion. However, our new allele with a 4.2-kb insertion reduced flowering in the low-latitude region, but produced unchanged flowering time in the high-latitude region relative to the 5.1-kb transposable insertion. Transcription analysis revealed that the new allele with 4.2-kb insertion versus the 5.1-kb insertion repressed and unchanged the transcription of ZmCCT in the low- and high-latitude regions, respectively. Thus, the allele with the 4.2-kb transposable insertion showed a completely opposite response to photoperiods between these two regions. Phylogenetic analysis revealed that the 4.2-kb transposable insertion in the two Northern flint corns originated from tropical maize. RNA-seq analysis and dual-luciferase transient expression assays further identified a conserved gene regulation network of ZmCCT between maize and rice, in which ZmCCT directly repressed the transcription of the florigen gene ZCN8 via ZmEhd1. Our results suggest that transposable elements play an important role in maize adaptation.

krn1, a major quantitative trait locus for kernel row number in maize.

Kernel row number is a fundamental component of maize (Zea mays) yield and an important target for maize breeding. The revolutionary transition from the two-rowed teosinte to maize with increased kernel row numbers dramatically enhanced yields during domestication. Kernel row number is controlled by many quantitative trait loci (QTLs), however most genes responsible for these QTLs remain uncharacterised and the molecular genetic mechanisms are unknown. Here, we combined map-based cloning and association mapping to identify a major QTL for kernel row number, krn1, which is likely to correspond to an existing gene (ids1/Ts6) encoding an AP2 domain protein homologous to the product of the wheat key domestication gene Q. The increased expression of ids1/Ts6 in two maize mutants increased spikelet pair meristem numbers and then enhanced kernel row numbers. Nucleotide diversity analysis further revealed that ids1/Ts6 and Q were under strong parallel selection in maize and wheat that increased their yields during domestication or improvement. RNA-seq revealed that ids1/Ts6 is involved in multiple pathways regulating spikelet pair meristem development, involving several key genes such as fea3, fea4 and ra3. The cloning of the krn1 gene will pave a new way to efficiently improve maize yield in the near future.

Parallel Domestication of the Heading Date 1 Gene in Cereals.

Flowering time is one of the key determinants of crop adaptation to local environments during domestication. However, the genetic basis underlying flowering time is yet to be elucidated in most cereals. Although staple cereals, such as rice, maize, wheat, barley, and sorghum, have spread and adapted to a wide range of ecological environments during domestication, it is yet to be determined whether they have a common genetic basis for flowering time. In this study, we show, through map-based cloning, that flowering time in sorghum is controlled by a major quantitative trait locus (QTL) Heading Date 1 (HD1), located on chromosome 10. The causal gene encodes the CONSTANS gene family which contains a CCT domain. A 5-bp deletion of a minor allele present in the coding sequence leads to a gene frameshift that delays flowering in sorghum. In contrast, in foxtail millet, association mapping of HD1 showed a common causal site with a splicing variant from "GT" to "AT" that was highly correlated with flowering time. In addition, the rice HD1 gene is known to harbor several causal variants controlling flowering time. These data indicate that the major flowering time QTL HD1 was under parallel domestication in sorghum, foxtail millet, and rice. The pattern of common mixed minor, or even rare, causal alleles in HD1 across different species may be representative of the genetic basis of the domestication syndrome. Furthermore, large DNA sequence analysis of HD1 revealed multiple origins for domesticated sorghum and a single origin for domesticated foxtail millet.

Gene duplication drove the loss of awn in sorghum.

Loss of the awn in some cereals, including sorghum, is a key transition during cereal domestication or improvement that has facilitated grain harvest and storage. The genetic basis of awn loss in sorghum during domestication or improvement remains unknown. Here, we identified the awn1 gene encoding a transcription factor with the ALOG domain that is responsible for awn loss during sorghum domestication or improvement. awn1 arose from a gene duplication on chromosome 10 that translocated to chromosome 3, recruiting a new promoter from the neighboring intergenic region filled with "noncoding DNA" and recreating the first exon and intron. awn1 acquired high expression after duplication and represses the elongation of awns in domesticated sorghum. Comparative mapping revealed high collinearity at the awn1 paralog locus on chromosome 10 across cereals, and awn growth and development were successfully reactivated on the rice spikelet by inactivating the rice awn1 ortholog. RNA-seq and DAP-seq revealed that as a transcriptional repressor, AWN1 bound directly to a motif in the regulatory regions of three MADS genes related to flower development and two genes, DL and LKS2, involved in awn development. AWN1 downregulates the expression of these genes, thereby repressing awn elongation. The preexistence of regulatory elements in the neighboring intergenic region of awn1 before domestication implicates that noncoding DNA may serve as a treasure trove for evolution during sorghum adaptation to a changing world. Taken together, our results suggest that gene duplication can rapidly drive the evolution of gene regulatory networks in plants.

Genetic Architecture of domestication- and improvement-related traits using a population derived from Sorghum virgatum and Sorghum bicolor.

The genetic basis of domestication and improvement remains largely unknown in sorghum as a typical multiple-origins species. In this study, the F2 and F3 populations derived from a cross between Sorghum virgatum and domesticated sorghum were used to study the genetic architecture of domestication- and improvement-related traits. We found that human selection had greatly reshaped sorghum through the Quantitative Trait Loci (QTLs) with large genetic effects in the traits of harvest, plant architecture and grain taste including the reduction of shattering, few branches, short plant stature and the removal of polyphenols from seed. The expansion of seed width was selected to improve the yield through accumulating small-effect QTLs. Two major QTLs of plant height (QTI-ph1 and dw1) were narrowed down into 24.5-kilobase (kb) and 13.9-kb, respectively. DNA diversity analysis and association mapping of dw1 gene suggested the functional variant (A1361 T) might originate from the same event not long time ago. Our results supported that parallel phenotypic changes across different species during domestication and improvement might share the same genetic basis, QTL × QTL interactions might not play an important role in the reshaping of traits during sorghum domestication and improvement, and offered new views on transgressive segregation and segregation distortion. Our study greatly deepens our understandings of the genetic basis of sorghum domestication and improvement.

The tin1 gene retains the function of promoting tillering in maize.

Sweet maize and popcorn retain tillering growth habit during maize diversification. However, the underlying molecular genetic mechanism remains unknown. Here, we show that the retention of maize tillering is controlled by a major quantitative trait locus (QTL), tin1, which encodes a C2H2-zinc-finger transcription factor that acts independently of tb1. In sweet maize, a splice-site variant from G/GT to C/GT leads to intron retention, which enhances tin1 transcript levels and consequently increases tiller number. Comparative genomics analysis and DNA diversity analysis reveal that tin1 is under parallel selection across different cereal species. tin1 is involved in multiple pathways, directly represses two tiller-related genes, gt1 and Laba1/An-2, and interacts with three TOPLESS proteins to regulate the outgrowth of tiller buds. Our results support that maize tin1, derived from a standing variation in wild progenitor teosinte population, determines tillering retention during maize diversification.