Associations between Early Thyroid-Stimulating Hormone Levels and Morbidities in Extremely Preterm Neonates.

INTRODUCTION: High-end cutoffs of thyroid-stimulating hormone (TSH) have been emphasized for hypothyroidism therapy in extremely preterm infants, but the significance of low TSH levels remains unknown. This study hypothesized that the spectrum of TSH levels by newborn screening after birth signifies specific morbidities in extremely preterm neonates. METHODS: The multicenter population cohort analyzed 434 extremely preterm neonates receiving TSH screening at 24-96 h of age in 2008-2019. Neonates were categorized by blood TSH levels into group 1: TSH <0.5 µU/mL, group 2: 0.5 ≤ TSH <2 µU/mL, group 3: 2 ≤ TSH <4 µU/mL, and group 4: TSH ≥4 µU/mL. Neonatal morbidities were categorized using the modified Neonatal Therapeutic Intervention Scoring System. RESULTS: The four groups differed in gestational age, birth weight, and the postnatal age at blood sampling so did the proportions of mechanical ventilation usage (p = 0.01), hypoxic respiratory failure (p = 0.005), high-grade intraventricular hemorrhage (p = 0.007), and periventricular leukomalacia (p = 0.048). Group 1 had higher severity scores for respiratory distress syndrome (RDS; effect size 0.39 [95% confidence interval [CI]: 0.18-0.59]) and brain injury (0.36 [0.15-0.57]) than group 2, which remained significant after adjusting for gestational age, birth weight, dopamine usage, and the postnatal age at TSH screening (RDS: mean + 0.45 points [95% CI: 0.11-0.79]; brain injury: +0.32 [0.11-0.54]). CONCLUSIONS: Low TSH levels in extremely preterm neonates are associated with severe RDS and brain injuries. Studies recruiting more neonates with complete thyroid function data are necessary to understand central-peripheral interactions of the hypothalamic-pituitary-thyroid axis.

The Saururus chinensis genome provides insights into the evolution of pollination strategies and herbaceousness in magnoliids.

Saururus chinensis, an herbaceous magnoliid without perianth, represents a clade of early-diverging angiosperms that have gone through woodiness-herbaceousness transition and pollination obstacles: the characteristic white leaves underneath inflorescence during flowering time are considered a substitute for perianth to attract insect pollinators. Here, using the newly sequenced S. chinensis genome, we revisited the phylogenetic position of magnoliids within mesangiosperms, and recovered a sister relationship for magnoliids and Chloranthales. By considering differentially expressed genes, we identified candidate genes that are involved in the morphogenesis of the white leaves in S. chinensis. Among those genes, we verified - in a transgenic experiment with Arabidopsis - that increasing the expression of the "pseudo-etiolation in light" gene (ScPEL) can inhibit the biosynthesis of chlorophyll. ScPEL is thus likely responsible for the switches between green and white leaves, suggesting that changes in gene expression may underlie the evolution of pollination strategies. Despite being an herbaceous plant, S. chinensis still has vascular cambium and maintains the potential for secondary growth as a woody plant, because the necessary machinery, i.e., the entire gene set involved in lignin biosynthesis, is well preserved. However, similar expression levels of two key genes (CCR and CAD) between the stem and other tissues in the lignin biosynthesis pathway are possibly associated with the herbaceous nature of S. chinensis. In conclusion, the S. chinensis genome provides valuable insights into the adaptive evolution of pollination in Saururaceae and reveals a possible mechanism for the evolution of herbaceousness in magnoliids.

Long-term outcomes of very early treated infantile-onset Pompe disease with short-term steroid premedication: experiences from a nationwide newborn screening programme.

BACKGROUND: Starting enzyme replacement therapy (ERT) before severe irreversible muscular damage occurs is important in infantile-onset Pompe disease (IOPD). This long-term follow-up study demonstrates our diagnostic and treatment strategies for IOPD and compares our clinical outcomes with those of other medical centres. METHODS: In this long-term follow-up study, we analysed the outcomes of very early ERT with premedication hydrocortisone in patients with IOPD. Out of 1 228 539 infants screened between 1 January 2010 and 28 February 2021, 33 newborns had confirmed IOPD in Taipei Veterans General Hospital. Twenty-six were regularly treated and monitored at Taipei Veterans General Hospital. Echocardiographic parameters, biomarkers, IgG antibodies against alglucosidase alpha, pulmonary function variables and developmental status were all assessed regularly over an average follow-up duration of 6.18±3.14 years. We compared the long-term treatment outcomes of our patients with those of other research groups. RESULTS: The average age at ERT initiation was 9.75±3.17 days for patients with classic IOPD. The average of the latest antialglucosidase alpha IgG titre was 669.23±1159.23. All enrolled patients had normal heart sizes, motor milestones, cognitive function and pulmonary function that were near-normal to normal. Compared with patients in other studies, our patients had better outcomes in all aspects. CONCLUSION: Very early ERT using our rapid diagnostic and treatment strategy enabled our patients with IOPD to have better outcomes than patients in other medical centres.

Liquid Chromatography-Tandem Mass Spectrometry in Newborn Screening Laboratories.

Tandem mass spectrometry (MS/MS) is the most universal platform currently available for the analysis of enzymatic activities and biomarkers in dried blood spots (DBS) for applications in newborn screening (NBS). Among the MS/MS applications in NBS, the most common is flow-injection analysis (FIA-) MS/MS, where the sample is introduced as a bolus injection into the mass spectrometer without the prior fractionation of analytes. Liquid chromatography combined with MS/MS (LC-MS/MS) has been employed for second-tier tests to reduce the false-positive rate associated with several nonspecific screening markers, beginning two decades ago. More recently, LC-MS/MS has been applied to primary screening for new conditions for which FIA-MS/MS or other methods, including genomic screening, are not yet adequate. In addition to providing a list of the currently used LC-MS/MS-based assays for NBS, the authors share their experience regarding the maintenance requirements of LC-MS/MS vs. FIA-MS/MS systems. The consensus is that the maintenance of LC-MS/MS and FIA-MS/MS instrumentation is similar, and LC-MS/MS has the advantage of allowing for a larger number of diseases to be screened for in a multiplex, cost-effective fashion with a high throughput and an adequate turnaround time.

Updated Confirmatory Diagnosis for Mucopolysaccharidoses in Taiwanese Infants and the Application of Gene Variants.

Mucopolysaccharidosis (MPS) is a lysosomal storage disease caused by genetic defects that result in deficiency of one specific enzyme activity, consequently impairing the stepwise degradation of glycosaminoglycans (GAGs). Except for MPS II, the other types of MPS have autosomal recessive inheritance in which two copies of an abnormal allele must be present in order for the disease to develop. In this study, we present the status of variant alleles and biochemistry results found in infants suspected of having MPS I, II, IVA, and VI. A total of 324 suspected infants, including 12 for MPS I, 223 for MPS II, 72 for MPS IVA, and 17 for MPS VI, who were referred for MPS confirmation from newborn screening centers in Taiwan, were enrolled. In all of these infants, one specific enzyme activity in dried blood spot filter paper was lower than the cut-off value in the first blood sample, as well asin a second follow-up sample. The confirmatory methods used in this study included Sanger sequencing, next-generation sequencing, leukocyte enzyme fluorometric assay, and GAG-derived disaccharides in urine using tandem mass spectrometry assays. The results showed that five, nine, and six infants had MPS I, II, and IVA, respectively, and all of them were asymptomatic. Thus, a laboratory diagnosis is extremely important to confirm the diagnosis of MPS. The other infants with identified nucleotide variations and reductions in leukocyte enzyme activities were categorized as being highly suspected cases requiring long-term and intensive follow-up examinations. In summary, the final confirmation of MPS depends on the most powerful biomarkers found in urine, i.e., the quantification of GAG-derived disaccharides including dermatan sulfate, heparan sulfate, and keratan sulfate, and analysis of genetic variants can help predict outcomes and guide treatment.

Newborn Screening Program for Mucopolysaccharidosis Type II and Long-Term Follow-Up of the Screen-Positive Subjects in Taiwan.

BACKGROUND: Mucopolysaccharidosis II (MPS II) is an X-linked disorder resulting from a deficiency in lysosomal enzyme iduronate-2-sulfatase (IDS), which causes the accumulation of glycosaminoglycans (GAGs) in the lysosomes of many tissues and organs, leading to progressive cellular dysfunction. An MPS II newborn screening program has been available in Taiwan since 2015. The aim of the current study was to collect and analyze the long-term follow-up data of the screen-positive subjects in this program. METHODS: From August 2015 to April 2022, 548,624 newborns were screened for MPS II by dried blood spots using tandem mass spectrometry, of which 202 suspected infants were referred to our hospital for confirmation. The diagnosis of MPS II was confirmed by IDS enzyme activity assay in leukocytes, quantitative determination of urinary GAGs by mass spectrometry, and identification of the IDS gene variant. RESULTS: Among the 202 referred infants, 10 (5%) with seven IDS gene variants were diagnosed with confirmed MPS II (Group 1), 151 (75%) with nine IDS gene variants were classified as having suspected MPS II or pseudodeficiency (Group 2), and 41 (20%) with five IDS gene variants were classified as not having MPS II (Group 3). Long-term follow-up every 6 months was arranged for the infants in Group 1 and Group 2. Intravenous enzyme replacement therapy (ERT) was started in four patients at 1, 0.5, 0.4, and 0.5 years of age, respectively. Three patients also received hematopoietic stem cell transplantation (HSCT) at 1.5, 0.9, and 0.6 years of age, respectively. After ERT and/or HSCT, IDS enzyme activity and the quantity of urinary GAGs significantly improved in all of these patients compared with the baseline data. CONCLUSIONS: Because of the progressive nature of MPS II, early diagnosis via a newborn screening program and timely initiation of ERT and/or HSCT before the occurrence of irreversible organ damage may lead to better clinical outcomes. The findings of the current study could serve as baseline data for the analysis of the long-term effects of ERT and HSCT in these patients.

Nationwide Newborn Screening Program for Mucopolysaccharidoses in Taiwan and an Update of the "Gold Standard" Criteria Required to Make a Confirmatory Diagnosis.

Mucopolysaccharidoses (MPSs) are a group of lysosomal storage diseases (LSDs) caused by an inherited gene defect. MPS patients can remain undetected unless the initial signs or symptoms have been identified. Newborn screening (NBS) programs for MPSs have been implemented in Taiwan since 2015, and more than 48.5% of confirmed cases of MPS have since been referred from these NBS programs. The purpose of this study was to report the current status of NBS for MPSs in Taiwan and update the gold standard criteria required to make a confirmative diagnosis of MPS, which requires the presence of the following three laboratory findings: (1) elevation of individual urinary glycosaminoglycan (GAG)-derived disaccharides detected by MS/MS-based assay; (2) deficient activity of a particular leukocyte enzyme by fluorometric assay; and (3) verification of heterogeneous or homogeneous variants by Sanger sequencing or next generation sequencing. Up to 30 April 2021, 599,962 newborn babies have been screened through the NBS programs for MPS type I, II, VI, and IVA, and a total of 255 infants have been referred to MacKay Memorial Hospital for a confirmatory diagnosis. Of these infants, four cases were confirmed to have MPS I, nine cases MPS II, and three cases MPS IVA, with prevalence rates of 0.67, 2.92, and 4.13 per 100,000 live births, respectively. Intensive long-term regular physical and laboratory examinations for asymptomatic infants with confirmed MPS or with highly suspected MPS can enhance the ability to administer ERT in a timely fashion.

Unraveling regulatory divergence, heterotic malleability, and allelic imbalance switching in rice due to drought stress.

The indica ecotypes, IR64, an elite drought-susceptible variety adapted to irrigated ecosystem, and Apo (IR55423-01 or NSIC RC9), a moderate drought-tolerant upland genotype together with their hybrid (IR64 × Apo) were exposed to non- and water-stress conditions. By sequencing (RNA-seq) these genotypes, we were able to map genes diverging in cis and/or trans factors. Under non-stress condition, cis dominantly explains (11.2%) regulatory differences, followed by trans (8.9%). Further analysis showed that water-limiting condition largely affects trans and cis + trans factors. On the molecular level, cis and/or trans regulatory divergence explains their genotypic differences and differential drought response. Between the two parental genotypes, Apo appears to exhibit more photosynthetic efficiency even under water-limiting condition and is ascribed to trans. Statistical analyses showed that regulatory divergence is significantly influenced by environmental conditions. Likewise, the mode of parental expression inheritance which drives heterosis (HET) is significantly affected by environmental conditions indicating the malleability of heterosis to external factors. Further analysis revealed that the HET class, dominance, was significantly enriched under water-stress condition. We also identified allelic imbalance switching in which several genes prefer IR64- (or Apo-) specific allele under non-stress condition but switched to Apo- (or IR64-) specific allele when exposed to water-stress condition.

Whole-genome microsynteny-based phylogeny of angiosperms.

Plant genomes vary greatly in size, organization, and architecture. Such structural differences may be highly relevant for inference of genome evolution dynamics and phylogeny. Indeed, microsynteny-the conservation of local gene content and order-is recognized as a valuable source of phylogenetic information, but its use for the inference of large phylogenies has been limited. Here, by combining synteny network analysis, matrix representation, and maximum likelihood phylogenetic inference, we provide a way to reconstruct phylogenies based on microsynteny information. Both simulations and use of empirical data sets show our method to be accurate, consistent, and widely applicable. As an example, we focus on the analysis of a large-scale whole-genome data set for angiosperms, including more than 120 available high-quality genomes, representing more than 50 different plant families and 30 orders. Our 'microsynteny-based' tree is largely congruent with phylogenies proposed based on more traditional sequence alignment-based methods and current phylogenetic classifications but differs for some long-contested and controversial relationships. For instance, our synteny-based tree finds Vitales as early diverging eudicots, Saxifragales within superasterids, and magnoliids as sister to monocots. We discuss how synteny-based phylogenetic inference can complement traditional methods and could provide additional insights into some long-standing controversial phylogenetic relationships.

Draft Genomes of Two Artocarpus Plants, Jackfruit (A. heterophyllus) and Breadfruit (A. altilis).

Two of the most economically important plants in the Artocarpus genus are jackfruit (A. heterophyllus Lam.) and breadfruit (A. altilis (Parkinson) Fosberg). Both species are long-lived trees that have been cultivated for thousands of years in their native regions. Today they are grown throughout tropical to subtropical areas as an important source of starch and other valuable nutrients. There are hundreds of breadfruit varieties that are native to Oceania, of which the most commonly distributed types are seedless triploids. Jackfruit is likely native to the Western Ghats of India and produces one of the largest tree-borne fruit structures (reaching up to 45 kg). To-date, there is limited genomic information for these two economically important species. Here, we generated 273 Gb and 227 Gb of raw data from jackfruit and breadfruit, respectively. The high-quality reads from jackfruit were assembled into 162,440 scaffolds totaling 982 Mb with 35,858 genes. Similarly, the breadfruit reads were assembled into 180,971 scaffolds totaling 833 Mb with 34,010 genes. A total of 2822 and 2034 expanded gene families were found in jackfruit and breadfruit, respectively, enriched in pathways including starch and sucrose metabolism, photosynthesis, and others. The copy number of several starch synthesis-related genes were found to be increased in jackfruit and breadfruit compared to closely-related species, and the tissue-specific expression might imply their sugar-rich and starch-rich characteristics. Overall, the publication of high-quality genomes for jackfruit and breadfruit provides information about their specific composition and the underlying genes involved in sugar and starch metabolism.

ORCAE-AOCC: A Centralized Portal for the Annotation of African Orphan Crop Genomes.

ORCAE (Online Resource for Community Annotation of Eukaryotes) is a public genome annotation curation resource. ORCAE-AOCC is a branch that is dedicated to the genomes published as part of the African Orphan Crops Consortium (AOCC). The motivation behind the development of the ORCAE platform was to create a knowledge-based website where the research-community can make contributions to improve genome annotations. All changes to any given gene-model or gene description are stored, and the entire annotation history can be retrieved. Genomes can either be set to "public" or "restricted" mode; anonymous users can browse public genomes but cannot make any changes. Aside from providing a user- friendly interface to view genome annotations, the platform also includes tools and information (such as gene expression evidence) that enables authorized users to edit and validate genome annotations. The ORCAE-AOCC platform will enable various stakeholders from around the world to coordinate their efforts to annotate and study underutilized crops.

The bracteatus pineapple genome and domestication of clonally propagated crops.

Domestication of clonally propagated crops such as pineapple from South America was hypothesized to be a 'one-step operation'. We sequenced the genome of Ananas comosus var. bracteatus CB5 and assembled 513 Mb into 25 chromosomes with 29,412 genes. Comparison of the genomes of CB5, F153 and MD2 elucidated the genomic basis of fiber production, color formation, sugar accumulation and fruit maturation. We also resequenced 89 Ananas genomes. Cultivars 'Smooth Cayenne' and 'Queen' exhibited ancient and recent admixture, while 'Singapore Spanish' supported a one-step operation of domestication. We identified 25 selective sweeps, including a strong sweep containing a pair of tandemly duplicated bromelain inhibitors. Four candidate genes for self-incompatibility were linked in F153, but were not functional in self-compatible CB5. Our findings support the coexistence of sexual recombination and a one-step operation in the domestication of clonally propagated crops. This work guides the exploration of sexual and asexual domestication trajectories in other clonally propagated crops.

Prediction of functional consequences of the five newly discovered G6PD variations in Taiwan.

Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency; OMIM #300908) is the most common inborn error disorders worldwide. While the G6PD is the key enzyme of removing oxidative stress in erythrocytes, the early diagnosis is utmost vital to prevent chronic and drug-, food- or infection-induced hemolytic anemia. The characterization of the mutations is also important for the subsequent genetic counseling, especially for female carrier with ambiguous enzyme activities and males with mild mutations. While multiplex SNaPshot assay and Sanger sequencing were performed on 500 G6PD deficient males, five newly discovered variations, namely c.187G > A (p.E63K), c.585G > C (p.Q195H), c.586A > T (p.I196F), c.743G > A (p.G248D), and c.1330G > A (p.V444I) were detected in the other six patients. These variants were previously named as the Pingtung, Tainan, Changhua, Chiayi, and Tainan-2 variants, respectively. The in silico analysis, as well as the prediction of the structure of the resultant mutant G6PD protein indicated that these five newly discovered variants might be disease causing mutations.

Applying a multiplexed primer extension method on dried blood spots increased the detection of carriers at risk of glucose-6-phosphate dehydrogenase deficiency in newborn screening program.

BACKGROUND: Patients with glucose-6-phosphate dehydrogenase deficiency might develop acute hemolytic anemia, chronic hemolytic anemia, and neonatal hyperbilirubinemia when exposed to high levels of oxidative stress. Severe hemolysis may occur in not only patients but also female carriers under certain conditions. However, 80%-85% of female carriers were undetected in an existing newborn screening program because of their wide-ranging levels of enzyme activity. METHODS: We developed a cost- and time-efficient multiplex SNaPshot assay using dried blood spots. RESULTS: By detecting 21 common mutations in Taiwan and Southeast Asia, the assay could determine 98.2% of the mutant alleles in our cohort of Taiwanese newborns. The 9 undetermined mutant alleles were consequently detected by Sanger sequencing, of which 5 unpublished variations-c.187G > A (Pingtung), c.585G > C (Tainan), c.586A > T (Changhua), c.743G > A (Chiayi), and c.1330G > A (Tainan-2)-were detected. Furthermore, 13% of mild mutations were missed in male infants whose enzyme levels at 6.1-7.0 U/gHb in the newborn screening program when set the cutoff value at 6.0 U/gHb. We therefore suggest increasing the cutoff value and applying the multiplex SNaPshot assay as the second tier for neonatal screening. CONCLUSIONS: Our approach could significantly increase the detection rate of male patients and female carriers with a reasonable cost and a reasonable number of clinic referrals.

Taiwan National Newborn Screening Program by Tandem Mass Spectrometry for Mucopolysaccharidoses Types I, II, and VI.

OBJECTIVE: To evaluate the initial cutoff values, rates of screen positives, and genotypes for the large-scale newborn screening program for multiple mucopolysaccharidoses (MPS) in Taiwan. STUDY DESIGN: More than 100 000 dried blood spots were collected consecutively as part of the national Taiwan newborn screening programs. Enzyme activities were measured by tandem mass spectrometry from dried blood spot punches. Genotypes were obtained when a second newborn screening specimen again had a decreased enzyme activity. Additional clinical evaluation was then initiated based on enzyme activity and/or genotype. RESULTS: Molecular genetic analysis for cases with low enzyme activity revealed 5 newborns with pathogenic alpha-L-iduronidase mutations, 3 newborns with pathogenic iduronate-2-sulfatase mutations, and 1 newborn was a carrier of an arylsulfatase B mutation. Several variants of unknown pathogenic significance were also identified, most likely causing pseudodeficiency. CONCLUSIONS: The highly robust tandem mass spectrometry-based enzyme assays for MPS-I, MPS-II, and MPS-VI allow for high-throughput newborn screening for these lysosomal storage disorders. Optimized cutoff values combined with second tier testing could largely eliminate false-positive results. Accordingly, newborn screening for these lysosomal storage disorders is possible.

Detecting 22q11.2 Deletion Syndrome in Newborns with Low T Cell Receptor Excision Circles from Severe Combined Immunodeficiency Screening.

OBJECTIVE: Based on experiences and results from newborn screening for severe combined immunodeficiency (SCID), we evaluated the occurrence of chromosome 22q11.2 deletion syndrome (22q11.2DS) in newborns with different T cell receptor excision circles (TREC) results and established a second tier genetic test for 22q11.2DS. STUDY DESIGN: Recalled dried blood spots from 486 newborns with TREC results <90 copies/uL were tested from the SCID newborn screening. Quantitative real-time polymerase chain reaction assay was used to detect the copy number of TBX1 and HIRA genes by simple DNA extraction method. Multiplex ligation dependent probe amplification was used for further confirmation. RESULTS: Four hundred sixty-eight cases were considered negative because their haploid copy number of TBX1 and HIRA genes was >0.75. Eighteen cases with TBX1 and/or HIRA gene copy number <0.75 were suspected as positive, and 13 cases were further confirmed with 22q11.2DS. Detection rates of 22q11.2DS were 10.7% (6/56) in TREC <30 copies, 6.8% (9/132) in <50 TREC copies, 4.6% (12/260) in <70 TREC copies, and 2.7% (13/486) in <90 TREC copies. CONCLUSIONS: 22q11.2DS detection can be incorporated into the second-tier assay in subjects with low TREC copies in SCID screening. The dried blood spot methods were feasible for 22q11.2DS newborn screening.

Publisher Correction: Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza.

This article was not made open access when initially published online, which was corrected before print publication. In addition, ORCID links were missing for 12 authors and have been added to the HTML and PDF versions of the article.

Insights into the Evolution of Multicellularity from the Sea Lettuce Genome.

We report here the 98.5 Mbp haploid genome (12,924 protein coding genes) of Ulva mutabilis, a ubiquitous and iconic representative of the Ulvophyceae or green seaweeds. Ulva's rapid and abundant growth makes it a key contributor to coastal biogeochemical cycles; its role in marine sulfur cycles is particularly important because it produces high levels of dimethylsulfoniopropionate (DMSP), the main precursor of volatile dimethyl sulfide (DMS). Rapid growth makes Ulva attractive biomass feedstock but also increasingly a driver of nuisance "green tides." Ulvophytes are key to understanding the evolution of multicellularity in the green lineage, and Ulva morphogenesis is dependent on bacterial signals, making it an important species with which to study cross-kingdom communication. Our sequenced genome informs these aspects of ulvophyte cell biology, physiology, and ecology. Gene family expansions associated with multicellularity are distinct from those of freshwater algae. Candidate genes, including some that arose following horizontal gene transfer from chromalveolates, are present for the transport and metabolism of DMSP. The Ulva genome offers, therefore, new opportunities to understand coastal and marine ecosystems and the fundamental evolution of the green lineage.

Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza.

The genus Oryza is a model system for the study of molecular evolution over time scales ranging from a few thousand to 15 million years. Using 13 reference genomes spanning the Oryza species tree, we show that despite few large-scale chromosomal rearrangements rapid species diversification is mirrored by lineage-specific emergence and turnover of many novel elements, including transposons, and potential new coding and noncoding genes. Our study resolves controversial areas of the Oryza phylogeny, showing a complex history of introgression among different chromosomes in the young 'AA' subclade containing the two domesticated species. This study highlights the prevalence of functionally coupled disease resistance genes and identifies many new haplotypes of potential use for future crop protection. Finally, this study marks a milestone in modern rice research with the release of a complete long-read assembly of IR 8 'Miracle Rice', which relieved famine and drove the Green Revolution in Asia 50 years ago.