Whole-genome and genome-wide association studies improve key agricultural traits of safflower for industrial and medicinal use

Safflower(Carthamus tinctorius)is widely cultivated around the world for its seeds and flowers.The presence of linoleic acid(LA)in its seeds and hydroxysafflor yellow A(HSYA)in its flowers are the crucial traits that enable safflower to be used for industrial and medicinal purposes.Understanding the genetic control of these traits is essential for optimizing the quality of safflower and its breeding.To further this research,we present a chromosome-scale assembly of the genome of the safflower variety‘Chuanhonghua 1’,which was achieved using an integrated strategy combining Illumina,Oxford Nanopore,and Hi-C sequencing.We obtained a 1.17-Gb assembly with a contig N50 of 1.08 Mb,and all assembled sequences were assigned to 12 pseudochromosomes.Safflower’s evolution involved the core eudicotγ-triplication event and a whole-genome duplication event,which led to large-scale genomic rearrangements.Extensive genomic shuffling has occurred since the divergence of the ancestor of dicotyledons.We conducted metabolite and transcriptome profiles with time-and part-dependent changes and screened candidate genes that significantly contribute to seed lipid biosynthesis.We also analyzed key gene families that participate in LA and HSYA biosynthesis.Additionally,we re-sequenced 220 safflower lines and carried out a genome-wide association study using high-quality SNP data for eight agronomic traits.We identified SNPs related to important traits in safflower.Besides,the candidate gene HH_034464(CtCGT1)was shown to be involved in the biosynthesis of HSYA.Overall,we provide a high-quality reference genome and elucidate the genetic basis of LA and HSYA biosynthesis in safflower.This vast amount of data will benefit further research for functional gene mining and breeding in safflower.

QTL Mapping of Isoflavone,Oil and Protein Contents in Soybean (Glycine max L.Merr.)

Soybean （Glycine max L. Merr.） is the world＇s foremost source of edible plant oil and proteins, meantime, the biologically active secondary metabolites such as saponins and isoflavones are benefit to human health. The objective of this study was to identify quantitative trait loci （QTL） and epistatic interactions associated with isoflavone, protein, and oil contents in soybean seeds. An F13 recombinant inbred line （RIL） comprising 474 lines was derived from a cross between Jindou 23 and Huibuzhi cultivars. SSR technique was employed for mapping of the QTLs. The QTLs for isoflavone, protein, and oil contents were analyzed and 23 QTLs were detected based on the constructed linkage map. Six QTLs for isoflavone content were localized in linkage groups J, N, D2, and G, eleven QTLs for oil content were localized in the linkage groups A1, A2, B2, C2, and D2, and six QTLs for protein content were localized in linkage groups B2, C2, G, and H1. The correlative analysis demonstrated that the isoflavone content had significant correlation with protein content, while significantly negative correlations was existed between oil and protein content, and significantly positive correlations was existed between protein and oil content. All these findings have laid an important basis for the marker assisted breeding in soybean. The phenotypic correlations of quantitative traits may be resulted from the correlation of the QTL controlling those traits.

Characterization and Expression Analysis of Four Glycine-Rich RNA-Binding Proteins Involved in Osmotic Response in Tobacco (Nicotiana tabacum cv. Xanthi)

Plants have developed many signals and specific genes＇ regulations at both transcriptional and post-transcriptional levels in order to tolerate and adapt to various environmental stresses. RNA-binding proteins （RBPs） play crucial roles in the post- transcriptional regulation via mRNA splicing, polyadenylation, sequence editing, transport, mRNA stability, mRNA localization, and translation. In this paper, four cDNAs of glycine-rich RNA-binding proteins （GR-RBPs）, named NtRGP-la, -lb, -2, and -3, were isolated from Nicotiana tabacum by RT-PCR analysis, and special emphases were given to the sequences alignment, phylogenetic analysis and gene expression. Sequences alignment revealed minor difference of cDNA sequences, but no difference of deduced proteins between N. sylvestris and N. tabacum. Phylogenetic alignment revealed that four cDNAs in tobacco were clustered into two different groups. NtRGP-2 and -3 were evolutionarily closest to Arabidopsis GR-RBPs genes and related to animal GR-RBPs genes, while NtRGP-la and -lb were closest to Gramineae GR-RBPs genes. The expression analyses of these four NtRGPs in response to different abiotic stresses revealed the similar expression pattern. Moreover, the four NtRGPs, especially NtRGP-la and NtRGP-3, were strongly induced by stresses including water, wound, cold, and high temperature, weakly induced by PEG, drought and SA, while reduced by NaC1 and unaffected by ABA treatment. The fact that all of these abiotic stresses included in our experiments affected the water balance and resulted in osmotic stress on cellular level, suggests that NtRGPs in tobacco should be a family of crucial osmosis-related proteins, and may play a key role in signal transduction with ABA-independent pathway under abiotic stresses.

Development and characterization of new allohexaploid resistant to web blotch in peanut

Peanut diseases seriously threaten peanut production, creating disease-resistant materials via interspecific hybridization is an effective way to deal with this problem. In this study, the embryo of an interspecific F1 hybrid was obtained by crossing the Silihong(Slh) cultivar with Arachis duranensis(ZW55), a diploid wild species. Seedlings were generated by embryo rescue and tissue culture. A true interspecific hybrid was then confirmed by cytological methods and molecular markers. After treating seedlings with colchicine during in vitro multiplication, the established interspecific F1 hybrid produced seeds which were named as Am1210. With oligonucleotide fluorescence in situ hybridization(Oligo FISH), molecular marker evaluations, morphological and web blotch resistance characterization, we found that: 1) Am1210 was an allohexaploid between Slh and ZW55;2) the traits of spreading lateral branches, single-seeded or double-seeded pods and red seed coats were observed to be dominant compared to the erect type, multiple-seeded pods and brown seed coats;3) the web blotch resistance of Am1210 was significantly improved than that of Slh, indicating the contribution of the web blotch resistance from the wild parent A. duranensis. In addition, 69 dominant and co-dominant molecular markers were developed which could be both used to verify the hybrid in this study and to identify translocation or introgression lines with A. duranensis chromosome fragments in future studies as well.

Physiological and anatomical changes in two rapeseed (Brassica napus L.) genotypes under drought stress conditions

Understanding the mechanisms of drought resistance in crop species is crucial for the selection and breeding of tolerant rapeseed(Brassica napus L.)varieties.The present study aimed to assess the physiological and anatomical responses of two rapeseed genotypes,P287(drought-tolerant)and T88(drought-sensitive)under three intensities of drought stress.All physiological and anatomical parameters related to drought acclimation were significantly altered in both genotypes under stress conditions.At the fourth-leaf stage,the relative water content,chlorophyll content,protein content,malondialdehyde content,and the activities of peroxidase and catalase in P287 were significantly higher than those in T88,particularly under severe drought conditions.After rehydration,all physiological indexes recovered rapidly,especially in P287.In addition,under drought stress,compared with T88,P287 had thicker palisade tissue,thinner spongy tissue,higher ratio of chloroplast length to chloroplast width,higher stomatal density and stomatal closure rate.Overall,the interaction between physiological and anatomical features improved the drought tolerance of P287 under drought stress conditions.

Chromosome painting of telomeric repeats reveals new evidence for genome evolution in peanut

Interspecific hybridization is an important approach to improve cultivated peanut varieties. Cytological markers such as tandem repeats will facilitate alien gene introgression in peanut. Telomeric repeats have also been frequently used in chromosome research. Most plant telomeric repeats are（TTTAGGG）n that are mainly distributed at the chromosome ends, although interstitial telomeric repeats（ITRs） are also commonly identified. In this study, the telomeric repeat was chromosomally localized in 10 Arachis species through sequential GISH（genomic in situ hybridization） and FISH（fluorescence in situ hybridization） combined with 4＇,6-diamidino-2-phenylindole（DAPI） staining. Six ITRs were identified such as in the centromeric region of chromosome Bi5 in Arachis ipa？nsis, pericentromeric regions of chromosomes As5 in A. stenosperma, Bho7 in A. hoehnei and Av5 in A. villosa, nucleolar organizer regions of chromosomes As3 in A. stenosperma and Adi3 in A. diogoi, subtelomeric regions of chromosomes Bho9 in A. hoehnei and Adu7 in A. duranensis, and telomeric region of chromosome Es7 in A. stenophylla. The distributions of the telomeric repeat, 5S r DNA, 45 S r DNA and DAPI staining pattern provided not only ways of distinguishing different chromosomes, but also karyotypes with a higher resolution that could be used in evolutionary genome research. The distribution of telomeric repeats, 5S r DNA and 45 S r DNA sites in this study, along with inversions detected on the long arms of chromosomes Kb10 and Bho10, indicated frequent chromosomal rearrangements during evolution of Arachis species.

Identification of a major QTL for Hessian fly resistance in wheat cultivar‘Chokwang’

The Hessian fly(HF,Mayetiola destructor)is one of the destructive pests of wheat(Triticum aestivum L.)worldwide.Resistant cultivars can effectively minimize wheat damage due to this insect pest.To identify new quantitative trait loci(QTL)for HF resistance,a population of recombinant inbred lines(RILs)was developed from a cross between the HF-resistant wheat cultivar‘Chokwang’and susceptible wheat‘Ning 7840’,and phenotyped for responses to HF attack.A linkage map was constructed using 1147 single nucleotide polymorphism(SNP)markers generated from genotyping-by-sequencing(GBS).One major QTL,QHf.hwwg-6 BS,for HF-resistance was identified on chromosome arm 6 BS,which explained up to84.0%of the phenotypic variation and was contributed by Chokwang.Two RILs showed recombination in the candidate interval of QHf.hwwg-6 BS,which delimited QHf.hwwg-6 BS to a 4.75 Mb physical interval between 6,028,601 bp and 10,779,424 bp on chromosome arm 6 BS of IWGSC Chinese Spring reference genome Ref Seq v2.0.Seven GBS-SNPs in the candidate interval were converted into Kompetitive allele specific polymerase chain reaction(KASP)markers.Two of them,KASP-6 B112698 and KASP-6 B7901215,were validated in a U.S.winter wheat panel.KASP-6 B112698 was nearly diagnostic,thus can be used to screen QHf.hwwg-6 BS and pyramid it with other resistance genes in breeding programs.

Molecular Diversity of a Seemingly Altitude Restricted <i>Ustilago kamerunensis</i>Isolates in Kenya: A Pathogen of Napier Grass

The Central region of Kenya which is the second largest market oriented dairy zone, faces a threat in milk production. The challenge is a disease known as the napier head smut caused by Ustilago kamerunensis. This fungal microorganism is a facultative pathogen which has been reported to cause yield losses in napier grass (Pennisetum purpureum) ranging from 25% to 46% across the affected areas. Additionally, there are reports of the continual spread of the disease into neighbouring county of Nakuru in Rift-Valley region which is the leading milk producing zone in the country. This scenario of spread is worrying combined with observation of variations in damage levels of napier grass clones across the five counties of Central Kenya. These observations led to the hypothesis that possible differences might be existing among the Ustilago kamerunensis variants in Kenya. Further, the differences in biomass yield losses that are within a certain percentage range mentioned-above, seemed to support the existence of possible differences. Therefore, to inform effective integrated management strategies of the pathogen in case it’s co-evolving, this study sought to determine the molecular differences of Ustilago kamerunensis isolates in affected counties using ITS 1 and 2 regions which are spanned by 5.8S ribosomal RNA gene. The Ustilago kamerunensis propagules were systematically collected from affected counties’ hot spot areas for sequencing and phylogenetic analysis. The study revealed the most affected areas to be within the mean altitude level of 1988.17 ± 71.97 metres above sea level. Further, differences in the growth in vitro and molecular characteristics of the seemingly altitude restricted isolates were observed. The Kiambu, Nyandarau and Nakuru counties isolates clustered together, whereas those of Murang’a, Nyeri and Kirinyaga formed another clade. The sequences of sixteen Ustilago kamerunensis isolates were deposited in GenBank with accession numbers ranging from MG722754 to MG722769. The results suggest the existence of possible genetic divergence of the isolates which might be reflected in their pathogenic potential too. Effective integration of management strategies is vital towards slowing the phenomenon for an optimal mitigation of the disease in Kenya.

Development of a rapid and efficient system for CR genes identification based on hairy root transformation in Brassicaceae

Many economically important crops and vegetables belonging to the cruciferous family are heavily endangered by clubroot disease caused by Plasmodiophora brassicae infection.Breeding of clubroot resistant cultivars based on mapping and cloning of resistant genes is commonly regarded as the most cost-effective and efficient way to fight against this disease.The traditional way of R gene functional validation requires stable transformation that is both time-and labor-consuming.In this study,a rapid and efficient hairy-root transgenic protocol mediated by Agrobacterium rhizogenes was developed.The transformation positive rate was over 80%in Brassica napus showed by GUS reporter gene and this transformation only took 1/6 of the time compared with stable transformation.The system was applicable to different B.napus varieties and other cruciferous crops including Brassica rapa and Brassica oleracea.In particular,two known CR genes,CRA3.7.1 and CRA8.2.4 were used respectively,as example to show that the system works well for CR gene study combined with subsequent P.brassicae infection in B.napus.Most importantly,it works both in over-expression that led to disease resistance,as well as in RNAi which led to disease susceptible phenotype.Therefore,this system can be used in batch-wise identification of CR genes,and also offered the possibility of manipulating key genes within the P.brassicae genome that could improve our knowledge on host-pathogen interaction.

The main restorer Rf3 of maize S type cytoplasmic male sterility encodes a PPR protein that functions in reduction of the transcripts of orf355

Dear Editor,Male sterile lines,especially cytoplasmic male sterile(CMS)lines,are extensively used in commercial hybrid seed production.Therefore a better understanding of the genetic basis of fertility restoration for CMS is important for the utility of heterosis in crops.In general,restorer of fertility(Rf)is controlled by one or two major genomic genes,and most of the Rf genes encode pro-teins containing P type pentatricopeptide repeat(PPR)motifs(Wang et al.,2006;Hu et al.,2012;Tang et al.,2014;Liu et al.,2016).S type CMS(CMS-S)is the main type of maize CMS with wide cytoplasmic sources,and orf355-orf77 in CMS-S was associated with male sterility(Zabala et al.,1997).The 5'stem-loop of the transcript of orf355-orf77 was thought to be associ-ated with its stability(Xiao et al.,2006).Recently,Xiao et al.(2020)confirmed that orf355 is the causal gene of CMS-S.Rf3,the main restorer of CMS-S,has been mapped to the long arm of chromosome 2 for a long time(Laughnan and Gabay,1978).Since then,great efforts have been made on genetic mapping and cloning of this gene(Xu et al.,2009).

Colonization of endophyte Acremonium sp. D212 in Panax notoginseng and rice mediated by auxin and jasmonic acid

Endophytic fungi can be beneficial to plant growth. However, the molecular mechanisms under-lying colonization of Acremonium spp. remain unclear. In this study, a novel endophytic Acremonium strain was isolated from the buds of Panax notoginseng and named Acremonium sp. D212. The Acremonium sp. D212 could colonize the roots of P. notoginseng, enhance the resistance of P. notoginseng to root rot disease, and promote root growth and saponin bio-synthesis in P. notoginseng. Acremonium sp. D212 could secrete indole-3-acetic acid (IAA) and jasmonic acid (JA), and inoculation with the fungus increased the endogenous levels of IAA and JA in P. noto-ginseng. Colonization of the Acremonium sp. D212 in the roots of the rice line Nipponbare was dependent on the concentration of methyl jasmonate (MeJA) (2–15μmol/L) and 1-naphthalenacetic acid (NAA) (10–20μmol/L). Moreover, the roots of the JA signaling-defective coi1-18 mutant were colonized by Acremonium sp. D212 to a lesser degree than those of the wild-type Nipponbare and miR393b-overexpressing lines, and the colonization was res-cued by MeJA but not by NAA. It suggests that the cross-talk between JA signaling and the auxin biosynthetic pathway plays a crucial role in the colonization of Acremonium sp. D212 in host plants.