A MITE variation-associated heat-inducible isoform of a heat-shock factor confers heat tolerance through regulation of JASMONATE ZIM-DOMAIN genes in rice.

High temperatures cause huge yield losses in rice. Heat-shock factors (Hsfs) are key transcription factors which regulate the expression of heat stress-responsive genes, but natural variation in and functional characterization of Hsfs have seldom been reported. A significant heat response locus was detected via a genome-wide association study (GWAS) using green leaf area as an indicative trait. A miniature inverted-repeat transposable element (MITE) in the promoter of a candidate gene, HTG3 (heat-tolerance gene on chromosome 3), was found to be significantly associated with heat-induced expression of HTG3 and heat tolerance (HT). The MITE-absent variant has been selected in heat-prone rice-growing regions. HTG3a is an alternatively spliced isoform encoding a functional Hsf, and experiments using overexpression and knockout rice lines showed that HTG3a positively regulates HT at both vegetative and reproductive stages. The HTG3-regulated genes were enriched for heat shock proteins and jasmonic acid signaling. Two heat-responsive JASMONATE ZIM-DOMAIN (JAZ) genes were confirmed to be directly upregulated by HTG3a, and one of them, OsJAZ9, positively regulates HT. We conclude that HTG3 plays an important role in HT through the regulation of JAZs and other heat-responsive genes. The MITE-absent allele may be valuable for HT breeding in rice.

Glycine max NNL1 restricts symbiotic compatibility with widely distributed bradyrhizobia via root hair infection.

Symbiosis between soybean (Glycine max) and rhizobia is essential for efficient nitrogen fixation. Rhizobial effectors secreted through the type-III secretion system are key for mediating the interactions between plants and rhizobia, but the molecular mechanism remains largely unknown. Here, our genome-wide association study for nodule number identified G. max Nodule Number Locus 1 (GmNNL1), which encodes a new R protein. GmNNL1 directly interacts with the nodulation outer protein P (NopP) effector from Bradyrhizobium USDA110 to trigger immunity and inhibit nodulation through root hair infection. The insertion of a 179 bp short interspersed nuclear element (SINE)-like transposon into GmNNL1 leads to the loss of function of GmNNL1, enabling bradyrhizobia to successfully nodulate soybeans through the root hair infection route and enhancing nitrogen fixation. Our findings provide important insights into the coevolution of soybean-bradyrhizobia compatibility and offer a way to design new legume-rhizobia interactions for efficient symbiotic nitrogen fixation.

Heterosis Derived From Nonadditive Effects of the BnFLC Homologs Coordinates Early Flowering and High Yield in Rapeseed (Brassica napus L.).

Early flowering facilitates crops to adapt multiple cropping systems or growing regions with a short frost-free season; however, it usually brings an obvious yield loss. In this study, we identified that the three genes, namely, BnFLC.A2, BnFLC.C2, and BnFLC.A3b, are the major determinants for the flowering time (FT) variation of two elite rapeseed (Brassica napus L.) accessions, i.e., 616A and R11. The early-flowering alleles (i.e., Bnflc.a2 and Bnflc.c2) and late-flowering allele (i.e., BnFLC.A3b) from R11 were introgressed into the recipient parent 616A through a breeding strategy of marker-assisted backcross, giving rise to eight homozygous near-isogenic lines (NILs) associated with these three loci and 19 NIL hybrids produced by the mutual crossing of these NILs. Phenotypic investigations showed that NILs displayed significant variations in both FT and plant yield (PY). Notably, genetic analysis indicated that BnFLC.A2, BnFLC.C2, and BnFLC.A3b have additive effects of 1.446, 1.365, and 1.361 g on PY, respectively, while their dominant effects reached 3.504, 2.991, and 3.284 g, respectively, indicating that the yield loss caused by early flowering can be successfully compensated by exploring the heterosis of FT genes in the hybrid NILs. Moreover, we further validated that the heterosis of FT genes in PY was also effective in non-NIL hybrids. The results demonstrate that the exploration of the potential heterosis underlying the FT genes can coordinate early flowering (maturation) and high yield in rapeseed (B. napus L.), providing an effective strategy for early flowering breeding in crops.

Soybean BARCSoySNP6K: An assay for soybean genetics and breeding research.

The limited number of recombinant events in recombinant inbred lines suggests that for a biparental population with a limited number of recombinant inbred lines, it is unnecessary to genotype the lines with many markers. For genomic prediction and selection, previous studies have demonstrated that only 1000-2000 genome-wide common markers across all lines/accessions are needed to reach maximum efficiency of genomic prediction in populations. Evaluation of too many markers will not only increase the cost but also generate redundant information. We developed a soybean (Glycine max) assay, BARCSoySNP6K, containing 6000 markers, which were carefully chosen from the SoySNP50K assay based on their position in the soybean genome and haplotype block, polymorphism among accessions and genotyping quality. The assay includes 5000 single nucleotide polymorphisms (SNPs) from euchromatic and 1000 from heterochromatic regions. The percentage of SNPs with minor allele frequency >0.10 was 95% and 91% in the euchromatic and heterochromatic regions, respectively. Analysis of progeny from two large families genotyped with SoySNP50K versus BARCSoySNP6K showed that the position of the common markers and number of unique bins along linkage maps were consistent based on the SNPs genotyped with the two assays; however, the rate of redundant markers was dramatically reduced with the BARCSoySNP6K. The BARCSoySNP6K assay is proven as an excellent tool for detecting quantitative trait loci, genomic selection and assessing genetic relationships. The assay is commercialized by Illumina Inc. and being used by soybean breeders and geneticists and the list of SNPs in the assay is an ideal resource for SNP genotyping by targeted amplicon sequencing.

Molecular mechanisms underpinning the multiallelic inheritance of MS5 in Brassica napus.

The Brassica-specific gene MS5 mediates early meiotic progression, and its allelic variants contribute to a valuable genic male sterility three-line hybrid production system in rapeseed (Brassica napus L.). However, the underlying mechanisms of its triallelic inheritance are poorly understood. Herein, we show that the restorer allele MS5a and the maintainer allele MS5c are both necessary for male fertility in B. napus. The functional divergence of MS5a and MS5c is strongly related to sequence variations in their coding regions and less strongly to their promoter regions. The male-sterile allele MS5b encodes a chimeric protein containing only the complete MS5 coiled-coil (CC) domain, having lost the MS5 superfamily domain. Both MS5a and MS5c can form homodimers in the nucleus via the CC domain. MS5b can interact competitively with MS5a or MS5c to form non-functional heterodimers. Owing to the close transcript levels of MS5b and MS5c in MS5b MS5c , these heterodimers induced a dominant-negative effect of MS5b on MS5c , resulting in a male-sterile phenotype. The extremely high transcript abundance of MS5a maintains sufficient MS5a homodimers in MS5a MS5b , causing the recovery of male sterility. These findings provide substantial genetic and molecular evidence to improve our understanding of the mechanisms underlying the multiallelic inheritance of MS5, and enable the construction of a solid foundation for improved use of the MS5-controlled GMS system in Brassica species.

Genetic Diversity and Phylogenetic Relationships of Annual and Perennial Glycine Species.

We have estimated the average genetic diversity of two Glycine annual and six perennial species based upon 76 orthologous gene sets and performed phylogenetic analysis, divergence analysis and tests for departure from neutrality of the eight species using 52 orthologous gene sets. In addition, 367 orthologous gene sets were used to estimate the relationships of 11 G. canescens accessions. Among the perennials, G. canescens showed the highest nucleotide diversity. The other perennials, except for G. tomentella, had higher nucleotide diversity than the two annuals. Phylogenetic analysis of the Glycine showed a similar genome grouping with the previous report except for G. cyrtoloba and G. stenophita which formed a sister clade in the study. Divergence analysis supported the phylogenetic relationships that G. falcata was the most divergent from G. max, followed by G. cyrtoloba, G. syndetika, G. tomentella D3, G. stenophita and G. canescens Most genic sequences were homogeneous in the levels of polymorphism and divergence between G. max and other Glycine species based on the HKA test, thus, Glycine perennials may have experienced a very similar evolution as inferred by trans-specific mutation analysis. The greater genetic diversity of most perennial Glycine species and their origins from the warmer and drier climates of Australia suggests the perennials maybe a potential source of heat and drought resistance that will be of value in the face of climate change.

Diversity and communities of culturable endophytic fungi from different tree peonies (geoherbs and non-geoherbs), and their biosynthetic potential analysis.

To know more about the potential roles of endophytic fungi in the formation mechanism of Daodi medicinal material, diversity and communities of culturable endophytic fungi in three types of tree peonies were investigated. Endophytic fungi of three types of tree peonies were isolated and identified. The diversity was analyzed. Bayesian trees constructed by MrBayes 3.2.6 after phylogenetic analysis of the ITS sequences. The endophytic fungi potential for synthesis of natural products was assessed by means of detecting NRPS and PKS gene sequences. In total, 364 endophytic fungi isolates representing 26 genera were recovered from Paeonia ostii 'Feng Dan', Paeonia ostii 'Luoyang Feng Dan', and Paeonia suffruticosa 'Luoyang Hong'. More culturable endophytic fungi appeared in P. suffruticosa 'Luoyang Hong' (206) compared with P. ostii 'Feng Dan' (60) and P. ostii 'Luoyang Feng Dan' (98). The fungal community of P. ostii 'Feng Dan' had the highest richness and diversity. PKSs and NRPS detection rates of endophytic fungi from P. ostii 'Feng Dan' are both the highest among the three types of tree peonies. Results indicate that endophytic fungus is an important factor of Daodi Cortex Moutan forming, and endophytic fungi in peony are related to genuineness of Cortex Moutan.

Overexpression of OsPGIP2 confers Sclerotinia sclerotiorum resistance in Brassica napus through increased activation of defense mechanisms.

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is the most serious disease affecting the yield of the agriculturally and economically important crop Brassica napus (rapeseed). In this study, Oryza sativa polygalacturonase-inhibiting protein 2 (OsPGIP2) was found to effectively enhanced rapeseed immunity against S. sclerotiorum infection. Leaf extracts of B. napus plants overexpressing OsPGIP2 showed enhanced S. sclerotiorum resistance by delaying pathogen infection. The constitutive expression of OsPGIP2 in rapeseed plants provided a rapid and effective defense response, which included the production of reactive oxygen species, interactions with S. sclerotiorum polygalacturonases (SsPG3 and SsPG6), and effects on the expression of defense genes. RNA sequencing analysis revealed that the pathogen induced many differentially expressed genes associated with pathogen recognition, redox homeostasis, mitogen-activated protein kinase signaling cascades, hormone signaling pathways, pathogen-/defense-related genes, and cell wall-related genes. The overexpression of OsPGIP2 also led to constitutively increased cell wall cellulose and hemicellulose contents in stems without compromising seed quality. The results demonstrate that OsPGIP2 plays a major role in rapeseed defense mechanisms, and we propose a model for OsPGIP2-conferred resistance to S. sclerotiorum in these plants.

Diversity and communities of culturable endophytic fungi from different tree peonies (geoherbs and non-geoherbs), and their biosynthetic potential analysis

Abstract To know more about the potential roles of endophytic fungi in the formation mechanism of Daodi medicinal material, diversity and communities of culturable endophytic fungi in three types of tree peonies were investigated. Endophytic fungi of three types of tree peonies were isolated and identified. The diversity was analyzed. Bayesian trees constructed by MrBayes 3.2.6 after phylogenetic analysis of the ITS sequences. The endophytic fungi potential for synthesis of natural products was assessed by means of detecting NRPS and PKS gene sequences. In total, 364 endophytic fungi isolates representing 26 genera were recovered from Paeonia ostii 'Feng Dan', Paeonia ostii 'Luoyang Feng Dan', and Paeonia suffruticosa 'Luoyang Hong'. More culturable endophytic fungi appeared in P. suffruticosa 'Luoyang Hong' (206) compared with P. ostii 'Feng Dan' (60) and P. ostii 'Luoyang Feng Dan' (98). The fungal community of P. ostii 'Feng Dan' had the highest richness and diversity. PKSs and NRPS detection rates of endophytic fungi from P. ostii 'Feng Dan' are both the highest among the three types of tree peonies. Results indicate that endophytic fungus is an important factor of Daodi Cortex Moutan forming, and endophytic fungi in peony are related to genuineness of Cortex Moutan.

Diversity and communities of culturable endophytic fungi from different tree peonies (geoherbs and non-geoherbs), and their biosynthetic potential analysis

Abstract To know more about the potential roles of endophytic fungi in the formation mechanism of Daodi medicinal material, diversity and communities of culturable endophytic fungi in three types of tree peonies were investigated. Endophytic fungi of three types of tree peonies were isolated and identified. The diversity was analyzed. Bayesian trees constructed by MrBayes 3.2.6 after phylogenetic analysis of the ITS sequences. The endophytic fungi potential for synthesis of natural products was assessed by means of detecting NRPS and PKS gene sequences. In total, 364 endophytic fungi isolates representing 26 genera were recovered from Paeonia ostii Feng Dan, Paeonia ostii Luoyang Feng Dan, and Paeonia suffruticosa Luoyang Hong. More culturable endophytic fungi appeared in P. suffruticosa Luoyang Hong (206) compared with P. ostii Feng Dan (60) and P. ostii Luoyang Feng Dan (98). The fungal community of P. ostii Feng Dan had the highest richness and diversity. PKSs and NRPS detection rates of endophytic fungi from P. ostii Feng Dan are both the highest among the three types of tree peonies. Results indicate that endophytic fungus is an important factor of Daodi Cortex Moutan forming, and endophytic fungi in peony are related to genuineness of Cortex Moutan.

A pentatricopeptide repeat protein restores nap cytoplasmic male sterility in Brassica napus.

Two forms of male-sterile cytoplasm, designated nap and pol, are found in the oilseed rape species, Brassica napus. The nap cytoplasm is observed in most B. napus varieties, and it confers male sterility on a limited number of cultivars that lack the corresponding restorer gene, Rfn. In the present study, using linkage analysis in combination with 5652 BC1 progeny derived from a cross between a nap cytoplasmic male sterility (CMS) line 181A and a restorer line H5, we delimited the Rfn gene to a 10.5 kb region on chromosome A09, which contained three putative ORFs. Complementation by transformation rescue revealed that the introduction of ORF2, which encodes a pentatricopeptide repeat (PPR) protein, resulted in the recovery of fertility of nap CMS plants. Expression analysis suggested that the Rfn was highly expressed in flower buds and it was preferentially expressed in the tapetum and meiocytes during anther development. Further RNA gel blots and immunodetection suggested that the Rfn gene may play a complicated role in restoring the nap CMS. Our work laid the foundation for dissecting the molecular basis of CMS fertility restoration and the nuclear-mitochondrial interactions in CMS/Rf systems.

Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP.

Predictable, clean genetic modification (GM) in livestock is important for reliable phenotyping and biosafety. Here we reported the generation of isozygous, functional myostatin (MSTN) knockout cloned pigs free of selectable marker gene (SMG) by CRISPR/Cas9 and Cre/LoxP. CRISPR/Cas9-mediated homologous recombination (HR) was exploited to knock out (KO) one allele of MSTN in pig primary cells. Cre recombinase was then used to excise the SMG with an efficiency of 82.7%. The SMG-free non-EGFP cells were isolated by flow cytometery and immediately used as donor nuclei for nuclear transfer. A total of 685 reconstructed embryos were transferred into three surrogates with one delivering two male live piglets. Molecular testing verified the mono-allelic MSTN KO and SMG deletion in these cloned pigs. Western blots showed approximately 50% decrease in MSTN and concurrent increased expression of myogenic genes in muscle. Histological examination revealed the enhanced myofiber quantity but myofiber size remained unaltered. Ultrasonic detection showed the increased longissimus muscle size and decreased backfat thickness. Precision editing of pig MSTN gene has generated isozygous, SMG-free MSTN KO cloned founders, which guaranteed a reliable route for elite livestock production and a strategy to minimize potential biological risks.

MS5 Mediates Early Meiotic Progression and Its Natural Variants May Have Applications for Hybrid Production in Brassica napus.

During meiotic prophase I, chromatin undergoes dynamic changes to establish a structural basis for essential meiotic events. However, the mechanism that coordinates chromosome structure and meiotic progression remains poorly understood in plants. Here, we characterized a spontaneous sterile mutant MS5(b)MS5(b) in oilseed rape (Brassica napus) and found its meiotic chromosomes were arrested at leptotene. MS5 is preferentially expressed in reproductive organs and encodes a Brassica-specific protein carrying conserved coiled-coil and DUF626 domains with unknown function. MS5 is essential for pairing of homologs in meiosis, but not necessary for the initiation of DNA double-strand breaks. The distribution of the axis element-associated protein ASY1 occurs independently of MS5, but localization of the meiotic cohesion subunit SYN1 requires functional MS5. Furthermore, both the central element of the synaptonemal complex and the recombination element do not properly form in MS5(b)MS5(b) mutants. Our results demonstrate that MS5 participates in progression of meiosis during early prophase I and its allelic variants lead to differences in fertility, which may provide a promising strategy for pollination control for heterosis breeding.

Map-based cloning reveals the complex organization of the BnRf locus and leads to the identification of BnRf(b), a male sterility gene, in Brassica napus.

KEY MESSAGE: Sequencing of BAC clones reveals the complex organization of the BnRf locus and allowed us to clone BnRf (b) , which encodes a nucleus-localized chimeric protein BnaA7.mtHSP70-1-like. The male sterility in an extensively used genic male sterility (GMS) line (9012A) in Brassica napus was regarded to be conferred by BnMs3/Bnms3 and the multiallelic BnRf locus including three alleles. We previously mapped BnRf to a 13.8 kb DNA fragment on the B. napus chromosome A7. In the present study, we isolated bacterial artificial chromosome clones individually covering the restorer allele BnRf (a) and the male-sterile allele BnRf (b) , and revealed that the candidate regions of BnRf (a) and BnRf (b) show complex structural variations relative to the maintainer allele BnRf (c). By analyzing the recombination events and the newly developed markers, we delimited BnRf (a) to a 35.9 kb DNA fragment that contained seven predicted open-reading frames (ORFs). However, genetic transformation of the ORF G14 from both the male-sterile and restorer lines into wild-type Arabidopsis plants led to a stable male-sterile phenotype matching a 9012A-derived GMS line (RG206A); moreover, the male sterility caused by G14 could be fully recovered by the restorer gene BnMs3. These facts indicate that BnRf (b) corresponds to G14 while BnRf (a) likely associates with another flanking ORF. G14 encodes a nucleus-localized chimeric protein designated as BnaA7.mtHSP70-1-like. Ectopic expression of G14 in Arabidopsis negatively regulates some vital genes responsible for tapetum degeneration, and delayed programmed cell death of tapetum and led to the developmental arrest of tetrads. Our work not only presents new insights on the hereditary model of sterility control but also lays a solid foundation for dissecting the molecular basis underlying male sterility and restoration in 9012A.

BnaC9.SMG7b Functions as a Positive Regulator of the Number of Seeds per Silique in Brassica napus by Regulating the Formation of Functional Female Gametophytes.

Number of seeds per silique (NSS) is an important determinant of seed yield potential in Brassicaceae crops, and it is controlled by naturally occurring quantitative trait loci. We previously mapped a major quantitative trait locus, qSS.C9, on the C9 chromosome that controls NSS in Brassica napus. To gain a better understanding of how qSS.C9 controls NSS in B. napus, we isolated this locus through a map-based cloning strategy. qSS.C9 encodes a predicted small protein with 119 amino acids, designated as BnaC9.SMG7b, that shows homology with the Ever ShorterTelomere1 tertratricopeptide repeats and Ever Shorter Telomere central domains of Arabidopsis (Arabidopsis thaliana) SUPPRESSOR WITH MORPHOGENETIC EFFECTS ON GENITALIA7 (SMG7). BnaC9.SMG7b plays a role in regulating the formation of functional female gametophyte, thus determining the formation of functional megaspores and then mature ovules. Natural loss or artificial knockdown of BnaC9.SMG7b significantly reduces the number of functional ovules per silique and thus, results in decreased seed number, indicating that qSS.C9 is a positive regulator of NSS in B. napus. Sequence and function analyses show that BnaC9.SMG7b experiences a subfunctionalization process that causes loss of function in nonsense-mediated mRNA decay, such as in Arabidopsis SMG7. Haplotype analysis in 84 accessions showed that the favorable BnaC9.SMG7b alleles are prevalent in modern B. napus germplasms, suggesting that this locus has been a major selection target of B. napus improvement. Our results represent the first step toward unraveling the molecular mechanism that controls the natural variation of NSS in B. napus.

Exploiting comparative mapping among Brassica species to accelerate the physical delimitation of a genic male-sterile locus (BnRf) in Brassica napus.

The recessive genic male sterility (RGMS) line 9012AB has been used as an important pollination control system for rapeseed hybrid production in China. Here, we report our study on physical mapping of one male-sterile locus (BnRf) in 9012AB by exploiting the comparative genomics among Brassica species. The genetic maps around BnRf from previous reports were integrated and enriched with markers from the Brassica A7 chromosome. Subsequent collinearity analysis of these markers contributed to the identification of a novel ancestral karyotype block F that possibly encompasses BnRf. Fourteen insertion/deletion markers were further developed from this conserved block and genotyped in three large backcross populations, leading to the construction of high-resolution local genetic maps where the BnRf locus was restricted to a less than 0.1-cM region. Moreover, it was observed that the target region in Brassica napus shares a high collinearity relationship with a region from the Brassica rapa A7 chromosome. A BnRf-cosegregated marker (AT3G23870) was then used to screen a B. napus bacterial artificial chromosome (BAC) library. From the resulting 16 positive BAC clones, one (JBnB089D05) was identified to most possibly contain the BnRf (c) allele. With the assistance of the genome sequence from the Brassica rapa homolog, the 13.8-kb DNA fragment covering both closest flanking markers from the BAC clone was isolated. Gene annotation based on the comparison of microcollinear regions among Brassica napus, B. rapa and Arabidopsis showed that five potential open reading frames reside in this fragment. These results provide a foundation for the characterization of the BnRf locus and allow a better understanding of the chromosome evolution around BnRf.