Comprehensive identification of major flowering time genes and their combinations,which determined rice distribution in Northeast China

Flowering time of rice (Oryza sativa L.) is among the most important agronomic traits for regional adaptation and grain yield. To date, a number of genes or quantitative trait loci (QTLs) controlling flowering time have been identified in rice, and diverse natural allelic variations for these flowering genes have been revealed, which suggested that the underlying regulation mechanism of flowering time in rice is very complicated. Northeast China is a major cultivation region for temperate japonica rice, where the temperature is cooler and the day length is longer. The regional adaptability of local rice cultivar is substantially different from that of other regions. Recently, some flowering genes have been proved to play roles in regulating flowering time of local cultivars. However, a comprehensive analysis of the effectiveness of these flowering genes has not been performed. In the present study, 395 cultivars collected from Northeast China is re-sequenced, SNP and InDel markers were called for 23 selected flowering-related genes. The heading date of these cultivars was also investigated for three consecutive years. Through association analysis, we found that Hd2, Hd4, and Hd5 are major flowering repressors, whereas Dth2 and Hd18 are major flowering promoters. Furthermore, Hd6 and Hd16 were identified as minor flowering repressors, and Hd17 was minor flowering promoter, in that their effectiveness can exclusively be detected when both Hd2 and Hd4 are functional. Collectively, we comprehensively identified the major and minor flowering genes which determine flowering time of temperate japonica rice grown in Northeast China.     

Combinations of Hd2 and Hd4 genes determine rice adaptability to Heilongjiang Province,northern limit of China

Heading date is a key trait in rice domestication and adaption, and a number of quantitative trait loci (QTLs) have been identified. The rice (Oryza sativa L.) cultivars in the Heilongjiang Province, t...     

Mapping of QTLs conferring extremely early heading in rice (Oryza sativa L.)

Two genes related to extremely early heading were identified in populations derived from crosses between Hoshinoyume, a variety adapted to the northernmost limit of rice cultivation (Hokkaido), and Nipponbare, a variety adapted to the temperate region of Japan. The segregations for heading date clearly revealed that a two-gene model determined the extremely early heading in the F₂ and BC₁F₁ populations under natural field conditions in Hokkaido. Using molecular markers corresponding to ten known quantitative trait loci (QTLs) for heading date, we carried out QTL analysis in the BC₁F₁ population and detected two QTLs, qDTH-7-1 and qDTH-7-2, both on chromosome 7, and observed epistatic interaction between them. We conclude that the recessive alleles of these two genes contribute to extremely early heading for the adaptation to Hokkaido environment and to stable rice production in Hokkaido. The relationships between the two QTLs identified in this study and known QTLs are discussed.     

Uncovering of major genetic factors generating naturally occurring variation in heading date among Asian rice cultivars

To dissect the genetic factors controlling naturally occurring variation of heading date in Asian rice cultivars, we performed QTL analyses using F₂ populations derived from crosses between a japonica cultivar, Koshihikari, and each of 12 cultivars originating from various regions in Asia. These 12 diverse cultivars varied in heading date under natural field conditions in Tsukuba, Japan. Transgressive segregation was observed in 10 F₂ combinations. QTL analyses using multiple crosses revealed a comprehensive series of loci involved in natural variation in flowering time. One to four QTLs were detected in each cross combination, and some QTLs were shared among combinations. The chromosomal locations of these QTLs corresponded well with those detected in other studies. The allelic effects of the QTLs varied among the cross combinations. Sequence analysis of several previously cloned genes controlling heading date, including Hd1, Hd3a, Hd6, RFT1, and Ghd7, identified several functional polymorphisms, indicating that allelic variation at these loci probably contributes to variation in heading date. Taken together, the QTL and sequencing results indicate that a large portion of the phenotypic variation in heading date in Asian rice cultivars could be generated by combinations of different alleles (possibly both loss- and gain-of-function) of the QTLs detected in this study.     

Hd1, Ghd7, and DTH8 synergistically determine the rice heading date and yield-related agronomic traits

Heading date determines the seasonal and regional adaptation of rice (Oryza sativa L.) varieties and is mainly controlled by photoperiod sensitivity (PS). The core heading date genes Hd1, Ghd7, DTH8, and PRR37 act synergistically in regulating the PS. In this study, we systematically analyze the heading date, PS, and agronomic traits of eight homozygous lines with various combinations of Hd1, Ghd7, and DTH8 alleles in the prr37 background under long-day (LD) and short-day (SD) conditions, respectively. We find that Hd1 alone promotes heading, regardless of the day length. However, under LDs, Hd1 suppresses flowering, in coordination with functional Ghd7 or with Ghd7 and DTH8. These loci cooperate to negatively regulate the Ehd1-Hd3a/RFT1 pathway and delay heading. Under SDs, Hd1 competes with various heading suppressors to promote heading. Therefore, the dual function of Hd1 is vital for PS. The lines carrying Hd1 alone show reduced plant height with fewer primary and secondary branches in panicles. Lines carrying Ghd7 and DTH8 (with hd1) show delayed heading and improve agronomic traits. Overall, our results reveal the regulation of rice PS flowering by the core heading date genes and their effects on agronomic traits, providing valuable information for the selection of rice varieties for adaptation to different light and temperature conditions.     

Historical changes in population structure during rice breeding programs in the northern limits of rice cultivation

Key message

The rice local population was clearly differentiated into six groups over the 100-year history of rice breeding programs in the northern limit of rice cultivation over the world.

Abstract

Genetic improvements in plant breeding programs in local regions have led to the development of new cultivars with specific agronomic traits under environmental conditions and generated the unique genetic structures of local populations. Understanding historical changes in genome structures and phenotypic characteristics within local populations may be useful for identifying profitable genes and/or genetic resources and the creation of new gene combinations in plant breeding programs. In the present study, historical changes were elucidated in genome structures and phenotypic characteristics during 100-year rice breeding programs in Hokkaido, the northern limit of rice cultivation in the world. We selected 63 rice cultivars to represent the historical diversity of this local population from landraces to the current breeding lines. The results of the phylogenetic analysis demonstrated that these cultivars clearly differentiated into six groups over the history of rice breeding programs. Significant differences among these groups were detected in five of the seven traits, indicating that the differentiation of the Hokkaido rice population into these groups was correlated with these phenotypic changes. These results demonstrated that breeding practices in Hokkaido have created new genetic structures for adaptability to specific environmental conditions and breeding objectives. They also provide a new strategy for rice breeding programs in which such unique genes in local populations in the world can explore the genetic potentials of the local populations.     

Genomic atlases of introgression and differentiation reveal breeding footprints in Chinese cultivated rice

The long history of cultivation and breeding has left a variety of footprints in the genomes of Asian cultivated rice (Oryza sativa L.). In this study, we focus on two types of genomic footprints, introgression and differentiation, in a population of more than 1200 Chinese rice accessions. We found that a Xian/indica and a temperate Geng/japonica accession respectively contained an average of 19.3-Mb and 6.8-Mb alien introgressed chromosomal segments, of which many contained functional sequence variants, quantitative trait loci, or genes controlling flowering, grain, and resistance traits. Notably, we found most introgressions, including the known heterotic loci Hd3a and TAC1, were distributed differentially between the female and male parents of three-line indica hybrid rice, indicating their potential contribution to heterosis. We also found many differentiated regions between subgroups within a subpopulation contained agronomically important loci, such as DTH7, Hd1 for heading date, and qCT7 for cold tolerance, providing new candidates for studying local adaptation or heterosis. Tracing these footprints allows us to better understand the genetic exchange or differentiation underlying agronomic traits in modern Chinese rice cultivars. These findings also provide potential targets for rice genetic research and breeding.     

Isolation of a major genetic interaction associated with an extreme phenotype using assorted F2 populations in rice

Detection of quantitative trait loci (QTLs) is dependent on the materials used in the analysis, as different combinations of parental materials may lead to different outcomes in QTLs for the same trait. On the other hand, an extreme phenotype associated with a given trait implies the potential involvement of a particular allele in various allelic interactions. A genetic factor associated with such an extreme phenotype may frequently be identified from various genetic populations consisting of different parental combinations. In this study, we attempted to uncover the genetic factor associated with extremely early heading date in rice, using various F2 populations. Heading date in rice has been characterized by at least 19 QTLs, from which 12 genes have been identified. A58, a rice strain with an extremely early heading date, is adapted to Hokkaido, the northernmost limit of rice cultivation. Six F2 populations derived from crosses of A58 with six other strains displayed a range of heading dates. Genotyping using 19 QTL markers indicated that the A58 allele of the Ghd7 locus was present in most F2 individuals exhibiting extremely early heading dates. This analysis also demonstrated that when the wild-type Ehd1 allele was present, the Ghd7 allele from A58 accelerated floral induction. The results of this study demonstrate that assorted F2 populations are valuable materials for comprehensive genotyping to explore major genetic factors for extreme phenotypes, and that this methodology is broadly applicable to other unknown traits.     

Accumulation of additive effects generates a strong photoperiod sensitivity in the extremely late-heading rice cultivar ‘Nona Bokra’

Many rice cultivars that originated from lower-latitude regions exhibit a strong photoperiod sensitivity (PS) and show extremely late heading under long-day conditions. Under natural day-length conditions during the cropping season in Japan, the indica rice cultivar ‘Nona Bokra’ from India showed extremely late heading (202 days to heading) compared to the japonica cultivar ‘Koshihikari’ (105 days), from Japan. To elucidate the genetic factors associated with such extremely late heading, we performed quantitative trait locus (QTL) analyses of heading date using an F₂ population and seven advanced backcross progeny (one BC₁F₂ and six BC₂F₂) derived from a cross between ‘Nona Bokra’ and ‘Koshihikari’. The analyses revealed 12 QTLs on seven chromosomes. The ‘Nona Bokra’ alleles of all QTLs contributed to an increase in heading date. Digenic interactions were rarely observed between QTLs. Based on the genetic parameters of the QTLs, such as additive effects and percentage of phenotypic variance explained, these 12 QTLs are likely generate a large proportion of the phenotypic variation observed in the heading dates between ‘Nona Bokra’ and ‘Koshihikari’. Comparison of chromosomal locations between heading date QTLs detected in this study and QTLs previously identified in ‘Nipponbare’ × ‘Kasalath’ populations revealed that eight of the heading date QTLs were recognized nearby the Hd1, Hd2, Hd3a, Hd4, Hd5, Hd6, Hd9, and Hd13. These results suggest that the strong PS in ‘Nona Bokra’ was generated mainly by the accumulation of additive effects of particular alleles at previously identified QTLs.     

Genetic interactions involved in the inhibition of heading by heading date QTL, <Emphasis Type="Italic">Hd2</Emphasis> in rice under long-day conditions

Heading date is the one of the most important traits in rice breeding, because it defines where rice can be cultivated and influences the expression of various agronomic traits. To examine the inhibition of heading by Heading date 2 (Hd2), previously detected on the distal end of chromosome 7’s long arm by quantitative trait locus (QTL) analysis, we developed backcross inbred lines (BILs) from Koshihikari, a leading Japanese cultivar, and Hayamasari, an extremely early heading cultivar. The BILs were cultivated under natural field conditions in Tsukuba Japan, and under long-day (14.5 h), extremely long-day (18 h), and short-day (10 h) conditions. Combinations of several QTLs near Hd1, Hd2, Ghd7, Hd5, and Hd16 were detected under these four conditions. Analysis of advanced backcross progenies revealed genetic interactions between Hd2 and Hd16 and between Hd2 and Ghd7. In the homozygous Koshihikari genetic background at Hd16, inhibition of heading by the Koshihikari allele at Hd2 was smaller than that with the Hayamasari Hd16 allele. Similarly, in the homozygous Koshihikari genetic background at Ghd7, the difference in heading date caused by different alleles at Hd2 was smaller than in plants homozygous for the Hayamasari Ghd7 allele. Based on these results, we conclude that Hd2 and its genetic interactions play an important role in controlling heading under long-day conditions. In addition, QTLs near Hd2, Hd16, and Ghd7, which are involved in inhibition of heading under long-day conditions, function in the same pathway that controls heading date.     

Genome-wide single nucleotide polymorphisms and insertion–deletions of Oryza sativa L. subsp. japonica cultivars grown near the northern limit of rice cultivation

Single nucleotide polymorphisms (SNPs) and insertions–deletions (InDels) are valuable molecular markers for molecular breeding among genetically closely related cultivars. Rice (Oryza sativa L. subsp. japonica) cultivars grown in Hokkaido (45–42°N), the northernmost region of rice paddy cultivation in Japan, have been bred for over 100 years for adaptation to low summer temperatures together with high yield and good eating quality. In this study, for 10 closely related rice cultivars released in Hokkaido and cultivar Koshihikari, we identified genome-wide SNPs and InDels by next-generation sequencing. More than 29 million reads from the Hokkaido cultivars, each 101 nucleotides long, were uniquely mapped to the Nipponbare reference genome. The average of the total nucleotide length of all uniquely mapped reads corresponded to 10.9 times (3,978 Mb with genome coverage of 90.7 %) the Nipponbare reference genome. An average of 99,955 putative SNPs (1.8 times the number in Koshihikari) and 14,617 putative InDels (also 1.8 times the number in Koshihikari) were detected in Hokkaido cultivars relative to the Nipponbare genome, which enabled analyses of the inheritance of pedigree haplotypes of four cultivars, SNPs and InDels among closely related Hokkaido cultivars, and haplotype blocks unique to Hokkaido cultivars. The comprehensive SNP and InDel data provide DNA marker resources and will facilitate quantitative trait locus analysis of biparental mapping of very closely related Hokkaido cultivars. Furthermore, the haplotype blocks unique to Hokkaido cultivars represent ideal genetic regions for improvement of cultivars to be grown near the northern and southern limits of rice cultivation.     

Detection of quantitative trait loci controlling extremely early heading in rice

To clarify the genetic basis of extremely early heading in rice, we conducted quantitative trait locus (QTL) analyses using F₂ populations from two genetically wide cross combinations, Hayamasari/Kasalath (HaF₂) and Hoshinoyume/Kasalath (HoF₂). Hayamasari and Hoshinoyume are extremely early-heading japonica cultivars. Photoperiod sensitivity is completely lost in Hayamasari and weak in Hoshinoyume. Three QTLs, QTL(chr6), QTL(chr7), and QTL(chr8), for days-to-heading (DTH) in HaF₂ were detected on chromosomes 6, 7, and 8, respectively, and QTL(chr6) and QTL(chr7) were detected in HoF₂. On the basis of the chromosomal locations, QTL(chr6), QTL(chr7), and QTL(chr8) may be likely to be Hd1, Hd4, and Hd5, respectively, which had been detected previously as QTLs for DTH in an F₂ population of Nipponbare × Kasalath. Alleles of QTL(chr7) decreased DTH dramatically in both Hayamasari and Hoshinoyume, suggesting that QTL(chr7) has a major role in determining extremely early heading. In addition, allele-specific interactions were detected between QTL(chr6), QTL(chr7) and QTL(chr8). This result suggests that not only allelic differences but also epistatic interactions contribute to extremely early heading. QTL(chr8) was detected in HaF₂, but not in HoF₂, suggesting that it determines the difference in DTH between Hayamasari and Hoshinoyume. A major QTL was also detected in the region of QTL(chr8) in QTL analysis using an F₂ population of Hayamasari × Hoshinoyume. This result supports the idea that QTL(chr8) is a major factor that determines the difference in DTH between Hayamasari and Hoshinoyume, and is involved in photoperiod sensitivity.     

Selection for low-temperature germinability on the short arm of chromosome 3 in rice cultivars adapted to Hokkaido,Japan

In plant breeding with intensive selection, the haplotype patterns in the targeted chromosomal regions may become monogenic among local populations with the most desirable combination of loci. This study demonstrated that the chromosomal region surrounding qLTG3-1 was under selection during rice breeding programs in a local region of Japan, Hokkaido. qLTG3-1 is a major quantitative trait loci controlling tolerance to low-temperature at the seed germination stage in rice, termed low-temperature germinability. A clear association between qLTG3-1 alleles and low-temperature germinability was detected among 64 rice cultivars from Hokkaido. The allele with a loss-of-function mutation seemed to be selected during rice breeding programs. Comparison of haplotype patterns along with the short arm of chromosome 3 revealed that the selection of qLTG3-1 alleles was focused on a distinct chromosomal region of at most 130 kb. In the short arm of chromosome 3, two major traits associated with the adaptability to local conditions have been identified; eating quality and heading date. This study demonstrated that recombinant haplotype patterns for these traits might shape the adaptability to local environmental conditions and market demands during rice breeding programs in addition to the selection of qLTG3-1 alleles. The present results provide new opportunities for the design of hybridization combinations based on the haplotype patterns of chromosomal regions under selection during rice breeding programs in local regions.     

Transformation of rice with the Arabidopsis floral regulator LEAFY causes early heading

Onset of flowering, or heading date, is an important agronomic trait of cereal crops such as rice and early-heading varieties are required for certain regions in which rice is cultivated. Since the floral control gene LEAFY from Arabidopsis can dramatically accelerate flowering in dictoyledonous plants, the usefulness of LEAFY for manipulating heading date in rice has been tested. Constitutive expression of LEAFY from the cauliflower mosaic virus 35S promoter caused early flowering in transgenic rice, with a heading date that was 26–34 days earlier than that of wild-type plants. Early flowering was accompanied by a small yield penalty and some panicle abnormality. These observations suggest that floral regulatory genes from Arabidopsis are useful tools for heading date improvement in cereal crops.     

Genomic adaptation of flowering‐time genes during the expansion of rice cultivation area

The diversification of flowering time in response to natural environments is critical for the spread of crops to diverse geographic regions. In contrast with recent advances in understanding the molecular basis of photoperiodic flowering in rice (Oryza sativa), little is known about how flowering‐time diversification is structured within rice subspecies. By analyzing genome sequencing data and a set of 429 chromosome segment substitution lines (CSSLs) originating from 10 diverse rice accessions with wide distributions, we revealed diverse effects of allelic variations for common flowering‐time quantitative trait loci in the recipient's background. Although functional variations associated with a few loci corresponded to standing variations among subspecies, the identified functional nucleotide polymorphisms occurred recently after rice subgroup differentiation, indicating that the functional diversity of flowering‐time gene sequences was not particularly associated with phylogenetic relationship between rice subspecies. Intensive analysis of the Hd1 genomic region identified the signature of an early introgression of the Hd1 with key mutation(s) in aus and temperate japonica accessions. Our data suggested that, after such key introgressions, new mutations were selected and accelerated the flowering‐time diversity within subspecies during the expansion of rice cultivation area. This finding may imply that new genome‐wide changes for flowering‐time adaptation are one of the critical determinants for establishing genomic architecture of local rice subgroups. In‐depth analyses of various rice genomes coupling with the genetically confirmed phenotypic changes in a large set of CSSLs enabled us to demonstrate how rice genome dynamics has coordinated with the adaptation of cultivated rice during the expansion of cultivation area.     

The Effect of the Crosstalk between Photoperiod and Temperature on the Heading-Date in Rice

Photoperiod and temperature are two important environmental factors that influence the heading-date of rice. Although the influence of the photoperiod on heading has been extensively reported in rice, the molecular mechanism for the temperature control of heading remains unknown. This study reports an early heading mutant derived from tissue culture lines of rice and investigates the heading-date of wild type and mutant in different photoperiod and temperature treatments. The linkage analysis showed that the mutant phenotype cosegregated with the Hd1 locus. Sequencing analysis found that the mutant contained two insertions and several single-base substitutions that caused a dramatic reduction in Hd1mRNA levels compared with wild type. The expression patterns of Hd1 and Hd3a were also analyzed in different photoperiod and temperature conditions, revealing that Hd1 mRNA levels displayed similar expression patterns for different photoperiod and temperature treatments, with high expression levels at night and reduced levels in the daytime. In addition, Hd1 displayed a slightly higher expression level under long-day and low temperature conditions. Hd3a mRNA was present at a very low level under low temperature conditions regardless of the day-length. This result suggests that suppression of Hd3a expression is a principle cause of late heading under low temperature and long-day conditions.     

Pleiotropism of the Photoperiod-Insensitive Allele of Hd1 on Heading Date,Plant Height and Yield Traits in Rice

Five populations segregated in isogenic backgrounds and three sets of near isogenic lines (NILs) overlapping in a 362.3-kb region covering heading date gene Hd1 were developed from the indica rice cross Zhenshan97 (ZS97)/Milyang 46 (MY46). They were used to analyze the effects of Hd1 on heading date, plant height and yield traits. In a background of the parental mixtures, the photoperiod-sensitive allele derived from ZS97 functioned in promoting and delaying flowering in the natural short-day and long-day conditions, respectively. In the background of ZS97, no response to the photoperiod was observed, whereas the photoperiod-insensitive allele derived from MY46 functioned in delaying flowering, increasing plant height, and enhancing grain productivity. The additive effects estimated in two NIL sets were 6.14 and 6.14 d for heading date, 4.46 and 5.55 cm for plant height, 10.82 and 11.54 for the number of spikelets per panicle, 6.82 and 8.00 for the number of grains per panicle, and 2.16 and 2.23 g for grain yield per plant, which explained 94.1% and 96.3%, 70.5% and 84.8%, 52.4% and 55.2%, 28.9% and 39.2%, and 36.5% and 26.9% of the phenotypic variances, respectively. Since the photoperiod-insensitive allele of Hd1 confers a long vegetative phase, it is a good candidate for breeding rice varieties with high yielding potential for low latitudes.