A rare Waxy allele coordinately improves rice eating and cooking quality and grain transparency

In rice (Oryza sativa), amylose content (AC) is the major factor that determines eating and cooking quality (ECQ). The diversity in AC is largely attributed to natural allelic variation at the Waxy (Wx) locus. Here we identified a rare Wx allele, Wx^mw, which combines a favorable AC, improved ECQ and grain transparency. Based on a phylogenetic analysis of Wx genomic sequences from 370 rice accessions, we speculated that Wx^mw may have derived from recombination between two important natural Wx alleles, Wxⁱⁿ and Wx^b. We validated the effects of Wx^mw on rice grain quality using both transgenic lines and near‐isogenic lines (NILs). When introgressed into the japonica Nipponbare (NIP) background, Wx^mw resulted in a moderate AC that was intermediate between that of NILs carrying the Wx^b allele and NILs with the Wx^mp allele. Notably, mature grains of NILs fixed for Wx^mw had an improved transparent endosperm relative to soft rice. Further, we introduced Wx^mw into a high‐yielding japonica cultivar via molecular marker‐assisted selection: the introgressed lines exhibited clear improvements in ECQ and endosperm transparency. Our results suggest that Wx^mw is a promising allele to improve grain quality, especially ECQ and grain transparency of high‐yielding japonica cultivars, in rice breeding programs.     

Novel Wx alleles generated by base editing for improvement of rice grain quality

Amylose content (AC), which is regulated by the Waxy (Wx) gene, is a major indicator of eating and cooking quality (ECQ) in rice (Oryza sativa). Thus far, only a limited number of mutations in the N-terminal domain of Wx were found to have a major impact on the AC of rice grains and no mutations with such effects were reported for other regions of the Wx protein. Here, nucleotide substitutions in the middle region of Wx were generated by adenine and cytosine base editors. The nucleotide substitutions led to changes in 15 amino acid residues of Wx, and a series of novel Wx alleles with ACs of 0.3%–29.43% (wild type with AC of 19.87%) were obtained. Importantly, the waxy^abe2 allele showed a “soft rice” AC, improved ECQ, favorable appearance, and no undesirable agronomic traits. The transgenes were removed from the waxy^abe2 progeny, generating a promising breeding material for improving rice grain quality.     

Analysis of intragenic recombination at wx in rice: correlation between the molecular and genetic maps within the locus.

In plant genomes as well as other eukaryotic genomes, meiotic recombination does not occur uniformly. At the level of the gene, high recombination frequencies are often observed within genetic loci in maize, but this feature of intragenic recombination is not seen at the csr1 locus in Arabidopsis. These observations suggest that meiotic recombination in plant genomes varies considerably among species. In the present study we investigated meiotic recombination at the wx locus in rice. The mutation sites of wx mutants induced by ethyl methanesulfonate (EMS) treatment or gamma-ray irradiation and a spontaneous wx mutant were physically characterized, and the genetic distances between those wx mutation sites were estimated by pollen analysis. Based on these results, the recombination frequency at the wx locus in rice was estimated as 27.3 kb/cM, which was about 10 times higher than the average for the genome, suggesting that there was a radically different rate of meiotic recombination for intra- and intergenic regions in the rice genome.     

CRISPR/Sc++-mediated genome editing in rice

Streptococcus canis Cas9 (ScCas9) is an RNA-guided endonuclease with NNG protospacer adjacent motif (PAM) specificity whose genome-editing activity in rice is locus-dependent. Here we investigated the performance of a ScCas9 variant named Sc⁺⁺ at different NNG PAM sites in the rice genome; Sc⁺⁺ harbors a T1227K mutation and the substitution of a positively charged loop (residues 367–376). Sc⁺⁺ nuclease achieved broader genome editing compared to the original ScCas9, and its nickase improved targeted base editing in transgenic rice plants. Using the high-efficiency adenine base editor rBE73b, we generated many new OsGS1 alleles suitable for screening of rice germplasm for potential herbicide resistance in the future. The CRISPR/Sc⁺⁺ system expands the genome-editing toolkit for rice.     

Seventeen Complementation Groups of Mutations Decreasing Meiotic Recombination in Schizosaccharomyces Pombe

We have analyzed 43 recessive mutations reducing meiotic intragenic recombination in Schizosaccharomyces pombe. These mutations were isolated by a screen for reduced plasmid-by-chromosome recombination at the ade6 locus. Sixteen of the mutations define 10 new complementation groups, bringing to 17 the number of genes identified to be involved in meiotic recombination. The mutations were grouped into three discrete classes depending on the severity of the recombination deficiency in crosses involving the ade6-M26 recombination hotspot. Class I mutations caused at least a 1000-fold reduction in M26-stimulated intragenic recombination at the ade6 locus. Class II mutations reduced M26-stimulated recombination approximately 100-fold. Class III mutations caused a 3-10-fold reduction in either M26-stimulated or non-hotspot recombination. We obtained multiple alleles of class I and class II mutations, suggesting that we may be nearing saturation for mutations of this type. As a first step toward mapping, we used mitotic segregation to assign fourteen of the rec genes to chromosomes. Mutations in the six rec genes tested also caused a decrease in intragenic recombination at the ura4 locus; five of these mutations also reduced intergenic recombination between the pro2 and arg3 genes. These results indicate that these multiple rec gene products are required for high level meiotic recombination throughout the S. pombe genome.     

Loss of Gn1a/OsCKX2 confers heavy-panicle rice with excellent lodging resistance

Significant achievements have been made in breeding programs for the heavy-panicle-type (HPT) rice (Oryza sativa) in Southwest China. The HPT varieties now exhibit excellent lodging resistance, allowing them to overcome the greater pressures caused by heavy panicles. However, the genetic mechanism of this lodging resistance remains elusive. Here, we isolated a major quantitative trait locus, Panicle Neck Diameter 1 (PND1), and identified the causal gene as GRAIN NUMBER 1A/CYTOKININ OXIDASE 2 (Gn1A/OsCKX2). The null gn1a allele from rice line R498 (gn1a^R498) improved lodging resistance through increasing the culm diameter and promoting crown root development. Loss-of-function of Gn1a/OsCKX2 led to cytokinin accumulation in the crown root tip and accelerated the development of adventitious roots. Gene pyramiding between the null gn1a^R498 allele with two gain-of-function alleles, STRONG CULM 2 (SCM2) and SCM3, further improved lodging resistance. Moreover, Gn1a/OsCKX2 had minimal influence on overall rice quality. Our research thus highlights the distinct genetic components of lodging resistance of HPT varieties and provides a strategy for tailor-made crop improvement of both yield and lodging resistance in rice.     

Genomic landscape of parallel domestication of upland rice and its implications

Parallel domestication has been widely acknowledged but itsgenetic basis remains largely unclear. As an important rice ecotype, upland rice was assumedly domesticated multiple times in two rice subspecies (Indica and Japonica) and provides a feasible system to explore the genetic basis of parallel domestication. To uncover the genome‐wide pattern of genetic differentiation between upland and lowland rice and explore the parallelism of genetic changes during upland rice domestication, we obtained whole‐genome sequences of 95 rice landraces and yielded genome‐wide expression data for five tissues of representative accessions of upland and lowland rice. Our phylogenetic analyses confirmed multiple domestications of the upland ecotype in two rice subspecies. Genomic scans based on resequencing data identified substantial differentiation between the upland and lowland ecotypes with 11.4% and 14.8% of the genome diverged between the two ecotypes in Indica and Japonica, respectively. Further genome‐wide gene expression analyses found that 30% of effectively expressed genes were significantly differentiated between two ecotypes, indicating the importance of regulation changes in the domestication of upland rice. Importantly, we found that only 1.8% of differentiated genomes and 1.6% of differentially expressed genes were shared by upland Indica and upland Japonica, suggestive of largely unparallel genetic alterations during upland rice domestication. These findings not only provide new insights into the genetic basis of parallel domestication at the genome scale but could also facilitate geneticimprovement and breeding of rice and crops in general.     

Pleiotropic effects of heterozygosity at the mating-type locus of the yeast Saccharomyces cerevisiae on repair, recombination and transformation

Sexual (MAT a/) and sexual (MAT a/a) strains of the yeast Saccharomyces cerevisiae, which are completely isogenic except at the MAT locus, were compared in their response to ultraviolet radiation. The effects of UV on survival, mitotic intragenic recombination, photoreactivation, and transformation efficiency with UV-irradiated plasmid DNA were examined. The sexual strain had enhanced survival and higher rates of mitotic intragenic recombination compared with the asexual strain. Exposure to visible light subsequent to irradiation increased the survival of both sexual and asexual strains, and decreased their rates of mitotic intragenic recombination. Similar results were obtained by Haladus and Zuk (1980) in their examination of sexual strains homozygous for rad6-1, and wild-type sexuals.

Our sexual strain was also consistently more proficient at transforming plasmid DNA, whether that DNA had been irradiated or not. When pre-irradiated with 25 J/m² of UV, MAT a/ cells transformed more efficiently than MAT a/a cells. When subsequently exposed to light, the ability of these pre-irradiated cells to transform decreased for both strains with increasing irradiation of the plasmid. A smaller decrease in transformation efficiency occurred when cells of both strains were kept in the dark.

When pre-irradiated with 100 J/m², the MAT a/ cells showed a 2-fold increase in their transformation efficiency of both irradiated and unirradiated plasmids by up to 2-fold, a phenomenon not seen in the MAT a/a cells even when pre-irradiated with much higher doses of UV. This increase in transformation efficiency was not, however, seen in the MAT a/ cells when they were exposed to visible light after UV irradiation. These results suggest that cells with the MAT a genotype have a UV-inducible system that increases the efficiency of transformation in the absence of visible light. This increase in transformation is not an induced increase in the repair of plasmid DNA, but rather an increase in the ability of pre-irradiated MAT a/ cells to take up exogenous DNA. MAT a/a cells do not appear to have a similarity inducible system. To the best of our knowledge, this phenomenon has not been previously reported.     

Molecular population genetic analysis of the streptokinase gene of Streptococcus pyogenes: mosaic alleles generated by recombination

To understand the mechanisms governing molecular evolution of the streptokinase gene (skn), a 384 bp DNA fragment encoding two variable regions of the molecule was characterized in 47 isolates of Streptococcus pyogenes. The results reveal that alleles of the streptokinase gene have a mosaic structure, and provide strong evidence for intragenic recombination. Moreover, organisms that are well differentiated in overall chromosomal character have identical skn alleles, which suggests that horizontal gene transfer and recombination have participated in the evolution of this locus. No simple relationship between skn allele and serum opacity factor production or specific disease was identified. The predicted amino acid sequences of highly divergent skn alleles are strikingly similar in hydrophobicity and hydrophobicity profiles, distribution of amphipathic and flexible regions, surface probability plots, and antigenic indices, indicating that despite extensive nucleotide polymorphism in the two skn variable regions, selective pressure has constrained overall structural divergence. These results add to an important emerging theme that intragenic recombination plays a critical role in diversifying genes coding for streptococcal virulence factors.     

Natural alleles of a uridine 5ʹ-diphospho-glucosyltransferase gene responsible for differential endosperm development between upland rice and paddy rice

Traditional upland rice generally exhibits insufficient grains resulting from abnormal endosperm development compared to paddy rice. However, the underlying molecular mechanism of this trait is poorly understood. Here, we cloned the uridine 5ʹ-diphospho (UDP)-glucosyltransferase gene EDR1 (Endosperm Development in Rice) responsible for differential endosperm development between upland rice and paddy rice by performing quantitative trait loci analysis and map-based cloning. EDR1 was highly expressed in developing seeds during grain filling. Natural variations in EDR1 significantly reduced the UDP-glucosyltransferase activity of EDR1^YZN compared to EDR1^YD1, resulting in abnormal endosperm development in the near-isogenic line, accompanied by insufficient grains and changes in grain quality. By analyzing the distribution of the two alleles EDR1^YD1 and EDR1^YZN among diverse paddy rice and upland rice varieties, we discovered that EDR1 was conserved in upland rice, but segregated in paddy rice. Further analyses of grain chalkiness in the alleles of EDR1^YD1 and EDR1^YZN varieties indicated that rice varieties harboring EDR1^YZN and EDR1^YD1 preferentially showed high chalkiness, and low chalkiness, respectively. Taken together, these results suggest that the UDP-glucosyltransferase gene EDR1 is an important determinant controlling differential endosperm development between upland rice and paddy rice.     

Mutations at the W locus affect survival of neural crest-derived melanocytes in the mouse

The development of melanoblasts in normally pigmented and dominant spotting (W) embryos was followed by in situ hybridisation to TRP-2/DT mRNA, which labels migratory melanoblasts from 10 days post coitum. Numerous melanoblasts migrate to the inner ear around 11 days. In contrast, few migratory melanoblasts are associated with the eye or skin at this stage and melanoblast distribution within the trunk and tail is patchy. The distribution of melanoblasts in 10.5–11-day-old W^v/W^v, W^sh/W^sh and W⁴¹/W⁴¹ mutants was similar to that in controls but melanoblast density was lower and by 12 days was severely reduced. These results suggest that mutations of the c-kit receptor tyrosine kinase encoded at the W locus do not alter early migration or differentiation of melanoblasts but severely affect melanoblast survival.     

Recombination Can Initiate and Terminate at a Large Number of Sites within the Rosy Locus of Drosophila Melanogaster

This report presents the results of a recombination experiment designed to question the existence of special sites for the initiation or termination of a recombination heteroduplex within the region of the rosy locus. Intragenic recombination events were monitored between two physically separated rosy mutant alleles ry301 and ry2 utilizing DNA restriction site polymorphisms as genetic markers. Both ry301 and ry2 are known from previous studies to be associated with gene conversion frequencies an order of magnitude lower than single site mutations. The mutations are associated with large, well defined insertions located as internal sites within the locus in prior intragenic mapping studies. On the molecular map, they represent large insertions approximately 2.7 kb apart in the second and third exons, respectively, of the XDH coding region. The present study monitors intragenic recombination in a mutant heterozygous genotype in which DNA homology is disrupted by these large discontinuities, greater than the region of DNA homology and flanking both sides of the locus. If initiation/or termination requires separate sites at either end of the locus, then intragenic recombination within the rosy locus of the heterozygote should be eliminated. Contrary to expectation, significant recombination between these sites is seen.     

Molecular evolution of the meiotic recombination pathway in mammals

Meiotic recombination shapes evolution and helps to ensure proper chromosome segregation in most species that reproduce sexually. Recombination itself evolves, with species showing considerable divergence in the rate of crossing‐over. However, the genetic basis of this divergence is poorly understood. Recombination events are produced via a complicated, but increasingly well‐described, cellular pathway. We apply a phylogenetic comparative approach to a carefully selected panel of genes involved in the processes leading to crossovers—spanning double‐strand break formation, strand invasion, the crossover/non‐crossover decision, and resolution—to reconstruct the evolution of the recombination pathway in eutherian mammals and identify components of the pathway likely to contribute to divergence between species. Eleven recombination genes, predominantly involved in the stabilization of homologous pairing and the crossover/non‐crossover decision, show evidence of rapid evolution and positive selection across mammals. We highlight TEX11 and associated genes involved in the synaptonemal complex and the early stages of the crossover/non‐crossover decision as candidates for the evolution of recombination rate. Evolutionary comparisons to MLH1 count, a surrogate for the number of crossovers, reveal a positive correlation between genome‐wide recombination rate and the rate of evolution at TEX11 across the mammalian phylogeny. Our results illustrate the power of viewing the evolution of recombination from a pathway perspective.     

稻属植物的基因组进化

水稻所在的稻属(Oryza)共有24个左右的物种。由于野生稻含有大量的优良农艺性状基因, 在水稻遗传学研究中日益受到重视。随着国际稻属基因组计划的开展, 越来越多的稻属基因组序列被测定, 稻属成为进行比较、功能和进化基因组学研究的模式系统。近期开展的一系列研究对稻属不同基因组区段以及全基因组序列的比较分析, 揭示了稻属在基因组大小、基因移动、多倍体进化、常染色质到异染色质的转化以及着丝粒区域的进化等方面的分子机制。转座子的活性以及转座子因非均等重组或非法重组而造成的删除, 对稻属基因组的扩增和收缩具有重要作用。DNA双链断裂修复介导的基因移动, 特别是非同源末端连接, 是稻属基因组非共线性基因形成的主要来源。稻属基因组从常染色质到异染色质的转换过程, 伴随着转座子的大量扩增、基因片段的区段性和串联重复以及从基因组其他位置不断捕获异染色质基因。对稻属不同物种间基因拷贝数、特异基因和重要农艺性状基因的进化等研究, 可揭示稻属不同物种间表型和适应性差异的分子基础, 将加速水稻的育种和改良。     

Recombination and the origin of sequence diversity in the DRB MHC class II locus in chamois (Rupicapra spp.)

We examined the evolutionary processes contributing to genetic diversity at the major histocompatibility complex (MHC) class II DRB locus in chamois (Rupicapra spp., subfamily Caprinae). We characterised the pattern of intragenic recombination (or homologous gene conversion) and quantified the amount of recombination in the genealogical history of the two chamois species, Pyrenean chamois (Rupicapra pyrenaica) and Alpine chamois (Rupicapra rupicapra). We found evidence for intragenic recombination, and the estimated amount of population recombination suggests that recombination has been a significant process in generating DRB allelic diversity in the genealogical history of the genus Rupicapra. Moreover, positive selection appears to act on the same peptide-binding residues in both analysed chamois species, but not in identical intensity. Recombination coupled with positive selection drives the rapid evolution at the peptide-binding sites in the MHC class II DRB gene. Many chamois MHC class II DRB alleles are thus much younger than previously assumed.     

High‐resolution insight into recombination events at the SD1 locus in rice

Recombination during meiosis plays an important role in genome evolution by reshuffling existing genetic variations into fresh combinations with the possibility of recovery of lost ancestral genotypes. While crossover (CO) events have been well studied, gene conversion events (GCs), which represent non‐reciprocal information transfer between chromosomes, are poorly documented and difficult to detect due to their relatively small converted tract size. Here, we document these GC events and their phenotypic effects at an important locus in rice containing the SD1 gene, where multiple defective alleles contributed to the semi‐dwarf phenotype of rice in the ‘Green Revolution’ of the 1960s. Here, physical separation of two defects allows recombination to generate the wild‐type SD1 gene, for which plant height can then be used as a reporter. By screening 18 000 F₂ progeny from a cross between two semi‐dwarf cultivars that carry these different defective alleles, we detected 24 GC events, indicating a conversion rate of ~3.3 × 10^?4 per marker per generation in a single meiotic cycle in rice. Furthermore, our data show that indels and single‐nucleotide polymorphisms (SNPs) do not differ significantly in GC rates, at least at the SD1 locus. Our results provide strong evidence that GC by itself can regain an ancestral phenotype that was lost through mutation. This GC detection approach is likely to be broadly applicable to natural or artificial alleles of other phenotype‐related functional genes, which are abundant in other plant genomes.     

Non-neutral evolution of the major histocompatibility complex class II gene DRB1 in the sac-winged bat Saccopteryx bilineata

The immune genes of the major histocompatibility complex (MHC) are classical examples for high levels of genetic diversity and non-neutral evolution. This is particularly true for the regions containing the antigen-binding sites as, for instance, in the exon 2 of the MHC class II gene DRB. We surveyed, for the first time in the order Chiroptera, the genetic diversity within this exon in the sac-winged bat Saccopteryx bilineata. We detected 11 alleles among 85 bats, of which 79 were sampled in one population. Pairwise comparisons revealed that interallelic sequence differences ranged between 3 and 22%, although nucleotide substitutions were not evenly distributed along the exon sequence. This was most probably the result of intragenic recombination. High levels of sequence divergence and significantly more nonsynonymous than synonymous substitutions (d(N)/d(S)>1) suggest long-term balancing selection. Thus, the data are consistent with the hypothesis that recombination gives rise to new alleles at the DRB locus of the sac-winged bat, and these are maintained in the population through balancing selection. In this respect, the sac-winged bat closely resembles other mammalian species.