Drought-triggered repression of miR166 promotes drought tolerance in soybean

Drought stress limits agricultural productivity worldwide.Identifying and characterizing genetic components of drought stress-tolerance networks may improve crop resistance to drought stress.We show that the regulatory module formed by miR166 and its target gene,ATHB14-LIKE,functions in the regulation of drought tolerance in soybean(Glycine max).Drought stress represses the accumulation of miR166,leading to upregulation of its target genes.Optimal knockdown of miR166 in the stable transgenic line GmSTTM166 conferred drought tolerance without affecting yield.Expression of ABA signaling pathway genes was regulated by the miR166-mediated regulatory pathway,and ATHB14-LIKE directly activates some of these genes.There is a feedback regulation between ATHB14-LIKE and MIR166 genes,and ATHB14-LIKE inhibits MIR166 expression.These findings reveal that drought-triggered regulation of the miR166-mediated regulatory pathway increases plants drought resistance,providing new insights into drought stress regulatory network in soybean.

Regulation of soybean stem growth habit:A ten-year progress report

Stem growth habit dictates plant architecture and influences flowering and podding(seed setting),making it an essential morphological and breeding agronomic trait of soybean(Glycine max).Stem growth habit in soybean is affected by photoperiod and environment and is determined by genetic variation at major genes.Classical genetic analysis identified two critical loci,designated Determinacy 1(Dt1)and Determinacy 2(Dt2).Dt1 is an ortholog of Arabidopsis thaliana TERMINAL FLOWER1(TFL1)and specifies an indeterminate stem growth habit,whereas Dt2 specifies a semi-determinate growth habit.MADS-box proteins,including Dt2,SUPPRESSOR OF OVEREXPRESSION OF CO1(GmSOC1)and MADS-box genes downregulated by E1(GmMDE),repress Dt1 expression.Photoreceptors encoded by the E3 and E4 loci regulate the expression of soybean FLOWERING LOCUS T(GmFT)orthologs via circadian clock genes and E1,and GmFTs compete with Dt1 to regulate stem growth habit.Study of the molecular mechanism underlying the regulation of stem growth habit in soybean has focused on the repression of Dt1 expression.Here we provide an overview of progress made in elucidating the genetic and molecular bases of stem growth habit in soybean,with emphasis on the molecular components responsible for integrating photoperiodic flowering and stem growth habit.

Characterization of dwarf mutants and molecular mapping of a dwarf locus in soybean

Plant height is one of the most important traits in soybean. The semi-dwarf soybean cultivars could improve the ability of lodging resistance to obtain higher yield. To broaden the dwarfism germplasm resources in soybean, 44 dwarf mutants were identified from a gamma rays mutagenized M-2 population. Two of these mutants, Gmdwf1（Glycine max dwarf 1） and Gmdwf2（Glycine max dwarf 2）, were investigated in this study. Genetic analysis showed that both mutants were inherited in a recessive manner and their mutated regions were delimited to a 2.610-Mb region on chromosome 1 by preliminary mapping. Further fine mapping study proved that the two mutants had a common deletion region of 1.552 Mb in the target region, which was located in a novel locus site without being reported previously. The dwarfism of Gmdwf1 could not be rescued by gibberellin（GA） and brassinolide（BR） treatments, which indicated that the biosynthesis of these hormones was not deficient in Gmdwf1.

Generation of male-sterile soybean lines with the CRISPR/Cas9 system

Soybean [Glycine max(L.) Merr.] provides a rich source of plant protein and oil worldwide. The commercial use of transgenic technology in soybean has become a classical example of the application of biotechnology to crop improvement. Although genetically modified soybeans have achieved commercial success,hybrid soybean breeding is also a potential way to increase soybean yield. Soybean cytoplasmic malesterile(CMS) lines have been used in three-line hybrid breeding systems, but their application to exploiting soybean heterosis has been limited by rare germplasm resource of sterile lines. The generation of various genetic diversity male-sterile soybean lines will help to overcome the shortcoming. In this study,we used targeted editing of AMS homologs in soybean by CRISPR/Cas9 technology for the first time to generate stable male-sterile lines. Targeted editing of GmAMS1 resulted in a male-sterile phenotype,while editing of GmAMS2 failed to produce male-sterile lines. GmAMS1 functions not only in the formation of the pollen wall but also in the controlling the degradation of the soybean tapetum.CRISPR/Cas9 technology could be used to rapidly produce stable male-sterile lines, providing new sterile-line materials for soybean hybrid breeding systems.

Identification of additional QTLs for flowering time by removing the effect of the maturity gene E1 in soybean

The adaptability of soybean to be grown at a wide range of latitudes is attributed to natural variation in the major genes and quantitative trait loci （QTLs） that control flowering time and maturity. Thus, the identification of genes controlling flowering time and maturity and the understanding of their molecular basis are critical for improving soybean productivity. However, due to the great effect of the major maturity gene E1 on flowering time, it is difficult to detect other small-effect QTLs. In this study, aiming to reduce the effect of the QTL, associated with the E1 gene, on the detection of other QTLs, we divided a population of 96 recombinant inbred lines （RILs） into two sub-populations： one with the E1 allele and another with the elns allele. Compared with the results of using all 96 recombinant inbred lines, additional QTLs for flowering time were identified in the sub-populations, two （qFT-B1 and qFT-H） in RILs with the E1 allele and one （qFT-J-2） in the RILs with the elnl allele, respectively. The three QTLs, qFT-B1, qFT-H and qFT-J-2 were true QTLs and played an important role in the regulation of growth period. Our data provides valuable information for the genetic mapping and gene cloning of traits controlling flowering time and maturity and will help a better understanding of the mechanism of photoperiod-regulated flowering and molecular breeding in soybean.

Multiplex CRISPR/Cas9-mediated knockout of soybean LNK2 advances flowering time

Flowering time is an important agronomic trait for soybean yield and adaptation. However, the genetic basis of soybean adaptation to diverse latitudes is still not clear. Four NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 2(LNK2) homeologs of Arabidopsis thaliana LNK2 were identified in soybean. Three single-guide RNAs were designed for editing the four LNK2 genes. A transgene-free homozygous quadruple mutant of the LNK2 genes was developed using the CRISPR(clustered regularly interspaced short palindromic repeats)/Cas9(CRISPR-associated protein 9). Under long-day(LD) conditions, the quadruple mutant flowered significantly earlier than the wild-type(WT). Quantitative real-time PCR(q RT-PCR)revealed that transcript levels of LNK2 were significantly lower in the quadruple mutant than in the WT under LD conditions. LNK2 promoted the expression of the legume-specific E1 gene and repressed the expression of FT2 a. Genetic markers were developed to identify LNK2 mutants for soybean breeding.These results indicate that CRISPR/Cas9-mediated targeted mutagenesis of four LNK2 genes shortens flowering time in soybean. Our findings identify novel components in flowering-time control in soybean and may be beneficial for further soybean breeding in high-latitude environments.

Development and validation of InDel markers for identification of QTL underlying flowering time in soybean

Soybean [Glycine max(L.) Merrill] is a major plant source of protein and oil. An accurate and well-saturated molecular linkage map is a prerequisite for forward genetic studies of gene function and for modern breeding for many useful agronomic traits. Next-generation sequence data available in public databases provides valuable information and offers new insights for rapid and efficient development of molecular markers. In this study, we attempted to show the feasibility and facility of using genomic resequencing data as raw material for identifying putative In Del markers. First, we identified 17,613 In Del sites among 56 soybean accessions and obtained 12,619 primer pairs. Second, we constructed a genetic map with a random subset of 2841 primer pairs and aligned 300 polymorphic markers with the 20 consensus linkage groups(LG). The total genetic distance was 2347.3 c M and the number of mapped markers per LG ranged from 10 to 23 with an average of 15 markers. The largest and smallest genetic distances between adjacent markers were 52.3 c M and 0.1 cM, respectively. Finally, we validated the genetic map constructed by newly developed In Del markers by QTL analysis of days to flowering(DTF) under different environments. One major QTL(qDTF4) and four minor QTL(qDTF20, qDTF13, qDTF12,and q DTF11) on 5 LGs were detected. These results demonstrate the utility of the In Del markers developed in this work for map-based cloning and molecular breeding in soybean.

QTL effects and epistatic interaction for flowering time and branch number in a soybean mapping population of Japanese×Chinese cultivars

Flowering time and branching type are important agronomic traits related to the adaptability and yield of soybean. Molecular bases for major flowering time or maturity loci, E1 to E4, have been identified. However, more flowering time genes in cultivars with different genetic backgrounds are needed to be mapped and cloned for a better understanding of flowering time regulation in soybean. In this study, we developed a population of Japanese cultivar（Toyomusume）×Chinese cultivar（Suinong 10） to map novel quantitative trait locus（QTL） for flowering time and branch number. A genetic linkage map of a F_2 population was constructed using 1 306 polymorphic single nucleotide polymorphism（SNP） markers using Illumina Soy SNP8 ki Select Bead Chip containing 7 189（SNPs）. Two major QTLs at E1 and E9, and two minor QTLs at a novel locus, qFT2_1 and at E3 region were mapped. Using other sets of F_2 populations and their derived progenies, the existence of a novel QTL of qFT2_1 was verified. qBR6_1, the major QTL for branch number was mapped to the proximate to the E1 gene, inferring that E1 gene or neighboring genetic factor is significantly contributing to the branch number.

High-Efficiency Reduction of Rice Amylose Content via CRISPR/Cas9-Mediated Base Editing

Rice is a staple food for more than half of the human population.It has been estimated that by 2030,rice production must increase by 40%to meet the growing demand(Khush,2005).In addition,with the improvement of people's living standards,the demand for elite rice with better eating and cooking quality(ECQ)is increasing.ECQ is determined by several factors,including amylose content(AC),gel consistency(GC),gelatinization temperature(GT)and viscosity,where AC is the predominant factor(Juliano,1998).

Genetic effects and plant architecture influences on outcrossing rate in soybean

Outcrossing rate is an important determinant of cytoplasmic male sterile(CMS)breeding and hybrid seed production for heterosis in soybean.Parental lines with a high outcrossing rate were screened for backcross breeding to obtain the high outcrossing rate maintenance B-lines and sterile A-lines.Application in production practices will help to increase hybrid soybean production.In this study,JLCMS82B and JLCMS89B were selected as parents for the construction of outcrossing rate segregation populations,and the progeny-array approach(PAA)and glyphosate resistant gene markers were used to determine outcrossing rates.We found that:(1)The outcrossing rate between JLCMS82B and JLCMS89B was significantly different;(2)the outcrossing rate of the F2 segregating populations was a quantitative trait,though whether an additive or epistatic effect exists required analysis with a triple test intersection analysis;(3)agronomic traits correlated with outcrossing rate;outcrossing rate was the highest with plant height of about 84 cm,lower number of plant branches,earlier flowering time,larger angle between the branches and the main stem,and with more divergent plant morphology.Correlation analysis between agronomic traits and outcrossing rate can effectively guide the screening of parents with a high outcrossing rate.

Elite sd1 alleles in japonica rice and their breeding applications in northeast China

The Green Revolution gene sd1 has been used extensively in modern rice breeding,especially in indica cultivars.However,elite sd1 alleles and related germplasm resources used for japonica rice breeding have not been identified,and extensive efforts are needed for japonica rice breeding to obtain new dwarfing sources.Data from MBKbase-Rice revealed seven sd1 haplotypes in indica and four in japonica rice.Two new sd1 alleles were identified in indica rice.In 295 japonica accessions from northeast Asia,except for the weak functional allele SD1-EQ,sd1-r was the major allele,reducing plant height in comparison with SD1-EQ.Japonica germplasm resources carrying reported sd1 alleles were identified by genotype searching and further verified by literature search,genealogical analysis,and d Caps markers.Pedigrees and geographic distribution showed that sd1-r is an excellent allele widely used in northern China and Tohoku in Japan,and sd1-j is commonly used in east China and Kyushu in Japan.Dongnong-and Xiushui-series cultivars carrying sd1-r and sd1-j,respectively,are essential branches of the backbone parents of Chinese japonica rice,Akihikari and Ce21,with the largest number of descendants and derived generations.In semi-dwarf japonica rice breeding,sd1-d was introgressed into Daohuaxiang 2(DHX2).Dwarf and semi-dwarf lines carrying sd1-d were selected and designated as 1279 and 1280,respectively,after withstanding typhoon-induced strong winds and heavy rains in 2020,and are anticipated to become useful intermediate materials for future genetic research and breeding.This work will facilitate the introduction,parental selection,and marker-assisted breeding,and provide a material basis for the next step in identifying favorable genes that selected together with the sd1 alleles in japonica backbone parents.

A critical role of PvFtsH2 in the degradation of photodamaged D1 protein in common bean

Light is required for initiating chloroplast biogenesis and photosynthesis;however,the photosystem II reaction center(PSII RC)can be photodamaged.In this study,we characterized pvsl1,a seedling-lethal mutant of Phaseolus vulgaris.This mutant showed lethality when exposed to sunlight irradiation and a yellow-green leaf phenotype when grown in a growth chamber under low-light conditions.We developed 124 insertion/deletion(INDEL)markers based on resequencing data of Dalong1 and PI60234,two local Chinese common bean cultivars,for genetic mapping.We identified Phvul.002G190900,which encodes the PvFtsH2 protein,as the candidate gene for this pvsl1 mutation through fine-mapping and functional analysis.A single-base deletion occurred in the coding region of Phvul.002G190900 in the pvsl1 mutant,resulting in a frameshift mutation and a truncated protein lacking the Zn^(2+)metalloprotease domain.Suppressed expression of Phvul.002G190900 at the transcriptional level was detected,while no change in the subcellular localization signal was observed.The seedlings of pvsl1 exhibited hypersensitivity to photoinhibition stress.In the pvsl1 mutant,abnormal accumulation of the D1 protein indicated a failure to rapidly degrade damaged D1 protein in the PSII RC.The results of this study demonstrated that PvFtsH2 is critically required for survival and maintaining photosynthetic activity by degrading photodamaged PSII RC D1 protein in common bean.

Molecular mechanisms for the photoperiodic regulation of flowering in soybean

Photoperiodic flowering is one of the most important factors affecting regional adaptation and yield in soybean(Glycine max). Plant adaptation to long-day conditions at higher latitudes requires early flowering and a reduction or loss of photoperiod sensitivity;adaptation to short-day conditions at lower latitudes involves delayed flowering, which prolongs vegetative growth for maximum yield potential. Due to the influence of numerous major loci and quantitative trait loci(QTLs), soybean has broad adaptability across latitudes. Forward genetic approaches have uncovered the molecular basis for several of these major maturity genes and QTLs. Moreover, the molecular characterization of orthologs of Arabidopsis thaliana flowering genes has enriched our understanding of the photoperiodic flowering pathway in soybean. Building on early insights into the importance of the photoreceptor phytochrome A, several circadian clock components have been integrated into the genetic network controlling flowering in soybean: E1, a repressor of FLOWERING LOCUS T orthologs, plays a central role in this network. Here, we provide an overview of recent progress in elucidating photoperiodic flowering in soybean, how it contributes to our fundamental understanding of flowering time control, and how this information could be used for molecular design and breeding of high-yielding soybean cultivars.

QNE1 is a key flowering regulator determining the length of the vegetative period in soybean cultivars

The soybean E1 gene is a major regulator that plays an important role in flowering time and maturity.However,it remains unclear how cultivars carrying the dominant E1 allele adapt to the higher latitudinal areas of northern China.We mapped the novel quantitative trait locus QNE1(QTL near E1) for flowering time to the region proximal to E1 on chromosome 6 in two mapping populations.Positional cloning revealed Glyma.06G204300,encoding a TCP-type transcription factor,as a strong candidate gene for QNE1.Association analysis further confirmed that functional single nucleotide polymorphisms(SNPs) at nucleotides 686 and 1,063 in the coding region of Glyma.06G204300 were significantly associated with flowering time.The protein encoded by the candidate gene is localized primarily to the nucleus.Furthermore,soybean and Brassica napus plants overexpressing Glyma.06G204300 exhibited early flowering.We conclude that despite their similar effects on flowering time,QNE1 and E4 may control flowering time through different regulatory mechanisms,based on expression studies and weighted gene co-expression network analysis of flowering time-related genes.Deciphering the molecular basis of QNE1 control of flowering time enriches our knowledge of flowering gene networks in soybean and will facilitate breeding soybean cultivars with broader latitudinal adaptation.

Parallel selection of distinct Tof5 alleles drove the adaptation of cultivated and wild soybean to high latitudes

Photoperiod responsiveness is a key factor limiting the geographic distribution of cultivated soybean and its wild ancestor.In particular,the genetic basis of the adaptation in wild soybean remains poorly understood.In this study,by combining whole-genome resequencing and genome-wide association studies we identified a novel locus,Time of Flowering 5(Tof5),which promotes flowering and enhances adaptation to high latitudes in both wild and cultivated soybean.By genomic,genetic and transgenic analyses we showed that Tof5 en-codes a homolog of Arabidopsis thaliana FRUITFULL(FUL).Importantly,further analyses suggested that different alleles of Tof5 have undergone parallel selection.The Tof5H1 allele was strongly selected by humans after the early domestication of cultivated soybean,while Tof5H2 allele was naturally selected in wild soybean,and in each case facilitating adaptation to high latitudes.Moreover,we found that the key flowering repressor E1 suppresses the transcription of Tof5 by binding to its promoter.In turn,Tof5 physically associates with the promoters of two important FLOWERING LOCUS T(FT),FT2a and FT5a,to upregulate their transcription and promote flowering under long photoperiods.Collectively,ourfindings provide insights into how wild soybean adapted to high latitudes through natural selection and indicate that cultivated soybean underwent changes in the same gene but evolved a distinct allele that was artificially selected after domestication.

High-efficiency breeding of early-maturing rice cultivars via CRISPR/Cas9-mediated genome editing

Rice is a staple food for more than half of the human population.It has been estimated that by 2030,40%more rice needs to be produced in order to meet the growing demand（Khush,2005）.One of the strategies to improve rice productivity is to enlarge rice growth areas, such as the northward expansion of the growth region in Heilongjiang Province, the northernmost region of China （Li et al., 2015）. However, the northward cultivation is accompanied with daylength extension and temperature decrease, which are unfavor- able for rice, a tropical short-day plant, to complete flowering and seed setting. Thus, the need for early-maturing rice cultivars with extremely low photoperiod sensitivity is urgent.

Agronomical selection on loss-of-function of GIGANTEA simultaneously facilitates soybean salt tolerance and early maturity

Salt stress and flowering time are major factors limiting geographic adaptation and yield productivity in soybean(Glycine max).Although improving crop salt tolerance and latitude adaptation are essential for efficient agricultural production,whether and how these two traits are integrated remains largely unknown.Here,we used a genome-wide association study to identify a major salt-tolerance locus controlled by E2,an ortholog of Arabidopsis thaliana GIGANTEA(GI).Loss of E2 function not only shortened flowering time and maturity,but also enhanced salt-tolerance in soybean.E2 delayed soybean flowering by enhancing the transcription of the core flowering suppressor gene E1,thereby repressing Flowering Locus T(FT)expression.An E2 knockout mutant e2^(CR) displayed reduced accumulation of reactive oxygen species(ROS)during the response to salt stress by releasing peroxidase,which functions in ROS scavenging to avoid cytotoxicity.Evolutionary and population genetic analyses also suggested that loss-of-function e2 alleles have been artificially selected during breeding for soybean adaptation to high-latitude regions with greater salt stress.Our findings provide insights into the coupled selection for adaptation to both latitude and salt stress in soybean;and offer an ideal target for molecular breeding of early-maturing and salt-tolerant cultivars.

A recent retrotransposon insertion of J caused E6 locus facilitating soybean adaptation into low latitude

Soybean(Glycine max) is an important legume crop that was domesticated in temperate regions.Soybean varieties from these regions generally mature early and exhibit extremely low yield when grown under inductive short-day(SD) conditions at low latitudes. The long-juvenile(LJ) trait, which is characterized by delayed flowering and maturity,and improved yield under SD conditions, allowed the cultivation of soybean to expand to lower latitudes. Two major loci control the LJ trait: J and E6. In the current study, positional cloning, sequence analysis, and transgenic complementation confirmed that E6 is a novel allele of J, the ortholog of Arabidopsis thaliana EARLY FLOWERING 3(ELF3). The mutant allele e6^(PG), which carries a Ty1/Copia-like retrotransposon insertion, does not suppress the legume-specific flowering repressor E1, allowing E1 to inhibit Flowering Locus T(FT) expression and thus delaying flowering and increasing yields under SD conditions. The e6^(PG)allele is a rare allele that has not been incorporated into modern breeding programs.The dysfunction of J might have greatly facilitated the adaptation of soybean to low latitudes. Our findings increase our understanding of the molecular mechanisms underlying the LJ trait and provide valuable resources for soybean breeding.

GmFtsH25 overexpression increases soybean seed yield by enhancing photosynthesis and photosynthates

Increasing plant photosynthetic capacity is a promising approach to boost yields,but it is particularly challenging in C3crops,such as soybean(Glycine max(L.)Merr.).Here,we identified GmFtsH25,encoding a member of the filamentation temperature-sensitive protein H protease family,as a major gene involved in soybean photosynthesis,using linkage mapping and a genome-wide association study.Overexpressing GmFtsH25 resulted in more grana thylakoid stacks in chloroplasts and increased photosynthetic efficiency and starch content,while knocking out GmFtsH25 produced the opposite phenotypes.GmFtsH25 interacted with photosystem I light harvesting complex 2(GmLHCa2),and this interaction may contribute to the observed enhanced photosynthesis.GmFtsH25 overexpression lines had superior yield traits,such as yield per plant,compared to the wild type and knockout lines.Additionally,we identified an elite haplotype of GmFtsH25,generated by natural mutations,which appears to have been selected during soybean domestication.Our study sheds light on the molecular mechanism by which GmFtsH25 modulates photosynthesis and provides a promising strategy for improving the yields of soybean and other crops.

A Strategy for the Acquisition and Analysis of Image-Based Phenome in Rice during the Whole Growth Period

As one of the most widely grown crops in the world,rice is not only a staple food but also a source of calorie intake for more than half of the world's population,occupying an important position in China's agricultural production.Thus,determining the inner potential connections between the genetic mechanisms and phenotypes of rice using dynamic analyses with high-throughput,nondestructive,and accurate methods based on high-throughput crop phenotyping facilities associated with rice genetics and breeding research is of vital importance.In this work,we developed a strategy for acquiring and analyzing 58 image-based traits(i-traits)during the whole growth period of rice.Up to 84.8%of the phenotypic variance of the rice yield could be explained by these i-traits.A total of 285 putative quantitative trait loci(QTLs)were detected for the i-traits,and principal components analysis was applied on the basis of the i-traits in the temporal and organ dimensions,in combination with a genome-wide association study that also isolated QTLs.Moreover,the differences among the different population structures and breeding regions of rice with regard to its phenotypic traits demonstrated good environmental adaptability,and the crop growth and development model also showed high inosculation in terms of the breeding-region latitude.In summary,the strategy developed here for the acquisition and analysis of image-based rice phenomes can provide a new approach and a different thinking direction for the extraction and analysis of crop phenotypes across the whole growth period and can thus be useful for future genetic improvements in rice.