Equine Warmblood fragile foal syndrome (WFFS) is a monogenetic defect of connective tissue with autosomal recessive inheritance. Foals born with WFFS manifest hyper‐extendible, abnormally thin, fragile skin that rips easily. WFFS is caused by the missense variant g.39927817C>T (rs1136065234; NC_009145.3, EquCab3.0) within the last exon of the PLOD1 (procollagen‐lysine, 2‐oxoglutarate 5‐dioxygenase 1) gene on equine chromosome 2. Using pedigree analyses and genotyping data of progeny, the potential origin of WFFS has been assumed to trace back to the English Thoroughbred stallion Dark Ronald (1905–1928). However, whether Dark Ronald, his father Bay Ronald or even an Arabian horse several hundred years earlier were actually the founders of the disorder remained elusive. WFFS belongs to a hereditary disease complex characterized by symptoms comprising skin hyperextensibility and abnormal fragility, hypermobility of joints, atrophic scarring and fragility of blood vessels, and was first described in humans as Ehlers–Danlos Syndrome.1 Ehlers–Danlos Syndrome‐like defects were also described in mammals in recent decades, e.g. cat,2 dog,2 rabbit,3 mink,4 cattle,5 sheep6 and horse.7 Based on pedigree data from 2000 WFFS tested horses the putative origin of the causative variant was traced back to Dark Ronald XX or his father Bay Ronald XX.8 Dark Ronald XX was a Thoroughbred stallion and started an early racing career in his home country then was sold into Germany in 1913. As a stud stallion in Graditz (Germany) and later in Altefeld (Germany), Dark Ronald XX heavily influenced the German Thoroughbred lines but his use in the German Warmblood breed had an equally long‐lasting effect. Even today, Dark Ronald XX is found in the pedigrees of many successful Thoroughbred and Warmblood sport horses in Germany. In 1928, suffering from colic, the horse was transported to the veterinary clinic of Halle University. After death, its remains (dehydrated and preserved skin, formalin‐fixed heart and skeleton) were preserved and today are still on display in the Museum of Domesticated Animals of the Central Natural Science Collections of Martin‐Luther‐University Halle‐Wittenberg (Fig. 1). In a recent report DNA extracted from a tooth sample from Dark Ronald has been used in a phylogenetic study.9 However, the deposited next‐generation sequencing files (European Nucleotide Archive ENA, study accession no. PRJEB31613, sample accession no. SAMEA5408260, run accession no. ERR3223826) show an extremely low coverage and unfortunately do not contain any reads of equine chromosome 2. Therefore, the genotype of Dark Ronald at the WFFS causative position (rs1136065234) remains unknown. However, the relicts enabled us to determine the genotype of Dark Ronald in somatic tissues.

For DNA extraction 2 cm² skin samples were used. PCR primers flanking rs1136065234 were designed using primer3 (forward primer, 5′‐AAACTGACGCTTCCTGTTGG‐3′; reverse primer, 5′‐CAGTTGTAACGCAGGAACCG‐3′).10 Primers were positioned to contain a high number of between‐species mismatches to prevent amplification of potential ancient or recent contaminations (Fig. S1). Amplicons (77 bp) were cloned into pGEM‐T vector, transformed into Escherichia coli XL1‐Blue and plasmid DNA preparations of 25 randomly selected positive colonies subsequently sequenced using the BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, Thermo Fisher Scientific GmbH). DNA sequences were separated on an ABI PRISM 3130xl Genetic Analyzer (Life Technologies) and compared with the equine PLOD1 reference sequence (NC_009145.3, EquCab3.0) using Sequencher 5.4.6 (Gene Codes Corporation). At the WFFS causative position g.39927817C>T all 25 clones showed the wt allele (Fig. 2). If this position was heterozygous in Dark Ronald one would expect approximately 50% of the clones to harbor the alternate allele. However, five polymorphic positions in intron 18 and one missense variant (g:39927814C>T) in exon 19 were detected (Table S1). Our analysis provides evidence that Dark Ronald was not the founder of the WFFS causative variant. Our findings are supported by a recent article showing that the WFFS‐causing variant most likely originates from a Hanoverian stallion born in 1861.11

马Warmblood脆性驹综合征（WFFS）是结缔组织的单基因缺陷，具有常染色体隐性遗传。WFFS出生的马驹表现出极度伸长，异常稀薄，易碎的皮肤。WFFS是由PLOD1的最后一个外显子中的错义变体g.39927817C> T（rs1136065234; NC_009145.3，EquCab3.0）引起的马染色体2上的（procollagen-lysine，2-oxoglutarate 5-dioxygenase 1）基因。使用系谱分析和后代的基因分型数据，假定WFFS的潜在起源可追溯到英国纯种马Dark Ronald（1905-1928） ）。但是，无论是黑罗纳德（Don Ronald），他的父亲罗纳德·罗纳德（Bay Ronald）还是几百年前的一匹阿拉伯马，实际上都是这种疾病的创始人都难以捉摸。WFFS属于遗传性疾病综合症，其特征是包括皮肤过度扩张和异常脆弱，关节活动过度，萎缩性瘢痕形成和血管脆弱等症状，并在人类中首次被描述为埃勒斯-丹洛斯综合症。1近几十年来，在哺乳动物中也描述了类似埃勒斯-丹洛斯综合症的缺陷，例如猫2狗，2只兔子，3只貂，4只牛，5只绵羊6和马。7根据2000个经过WFFS测试的马的血统数据，推定的致病性起源可追溯到Dark Ronald XX或他的父亲Bay Ronald XX。8Dark Ronald XX是纯种马，在他的祖国开始了早期的赛车生涯，然后于1913年出售给德国。作为Graditz（德国）和后来在Altefeld（德国）的种马，Dark Ronald XX对德国纯种马产生了重大影响。系，但他在德国Warmblood犬种中的使用同样具有长效作用。即使在今天，在德国许多成功的纯种和温血运动马的血统书中也发现了Dark Ronald XX。1928年，由于患肠绞痛，这匹马被运送到哈雷大学的兽医诊所。死后，其遗物（脱水和保存的皮肤，福尔马林固定的心脏和骨骼）得以保存，今天仍在马丁·路德·大学·哈雷·威登伯格自然科学收藏中心的家养动物博物馆中展出（图。 1）。9但是，存放的下一代测序文件（欧洲核苷酸档案馆ENA，研究登录号PRJEB31613，样品登录号SAMEA5408260，运行登录号ERR3223826）显示极低的覆盖率，不幸的是不包含任何马2号染色体的读数因此，在WFFS病因位置（rs1136065234）的Dark Ronald基因型仍然是未知的。但是，这些遗物使我们能够确定体细胞组织中暗黑罗纳德的基因型。

为了提取DNA ，使用2cm ^2的皮肤样品。rs1136065234侧翼的PCR引物使用引物3设计（正向引物5'-AAACTGACGCTTCCTGTTGG-3';反向引物5'-CAGTTGTAACGCAGGAACCG-3'）。放置了10个引物，使其在种间之间存在大量错配，以防止潜在的古老或近期污染扩大（图S1）。将扩增子（77 bp）克隆到pGEM-T载体中，并转化到大肠杆菌中随后使用BigDye Terminator 3.1版循环测序试剂盒（Applied Biosystems，Thermo Fisher Scientific GmbH）对25个随机选择的阳性菌落的XL1-Blue和质粒DNA制剂进行测序。在ABI PRISM 3130xl遗传分析仪（Life Technologies）上分离DNA序列，并与马PLOD1进行比较使用Sequencher 5.4.6（Gene Codes Corporation）的参考序列（NC_009145.3，EquCab3.0）。在WFFS的致病位g.39927817C> T，所有25个克隆均显示出wt等位基因（图2）。如果该位置在《黑暗罗纳德》中是杂合的，则可以预期约有50％的克隆包含替代等位基因。然而，在外显子19中检测到内含子18中的五个多态性位置和一个错义变体（g：39927814C> T）（表S1）。我们的分析提供了证据，证明Dark Ronald不​​是WFFS因果变体的创始人。最近的一篇文章支持了我们的发现，该文章表明，导致WFFS的变体最有可能起源于1861年出生的汉诺威种马。11