A mitochondrial DNA SNP is maternally inherited in hops (Humulus lupulus, L.) and segregates varieties of the renowned Brewer's Gold female lineage from the others

As revised in 2013 by Yang et al. [1], Humulus is one of the ten genera of the Cannabaceae family. The three Humulus species, whose cradle is Eastern Asia, are H. japonicus, Siebold & Zuccarini (or H. scandens, Loureiro), H. lupulus, Linné, known as hop, and H. yunnanensis, Hu.

H. lupulus, a dioecious diploid perennial plant was distributed primarily in Northern temperate parts of America, Asia and Europe. Its 2.57 Gbp [2] genome is composed of 20 chromosomes (2n=2x=20), of which one pair is recognised as sexual (XXfor female and XYfor male). Hops also exist as aneuploid, monoecious, triploid or tetraploid individuals [3]. The main use of hops since the Middle Age and up to now is as a “spice” in brewing, bringing bitterness, aroma and flavour to beer [4-6]. Only female plants are cultivated for their inflorescences, called cones (or strobili), which host lupulin glands containing the wanted specific resins. Their principal fraction, the -acids (humulone), which is the traditional basis of the hop commercial value, contributes, along with -acids (lupulone), to beer bitterness whereas its aroma and flavour are due to cone essential oils [6]. These remarkable metabolites are mostly synthesized from the isopentenyl pyrophosphate (IPP) by involving nuclear, plastidial and/or mitochondrial pathways [7, 8].

From germplasm collections, hosted in a few places dedicated to H. lupulus in producing countries, selected female and male individuals, used for controlled or open pollination, are requested inbreeding programs to generate and establish genetically stable new cultivars. Once such a potent new variety is chosen for its properties, it is vegetatively propagated in glasshouses and the resulting developed plantlets are positioned in the prepared gardens for further growth. Two to four-year trials in various places allow to establish its characteristics by both qualitative and quantitative studies, including the ease to cultivate and harvest, the resistance to diseases, the crop yield, sensory evaluations and biochemical analyses. Parental plants used inbreeding programs can be already existing varieties (mother plants) and males originating from previous crosses or wild type hops [9-11]. In hop gardens, and for each cultivated plant, up to 4 bines from annual shoots are fixed during Spring on a trellis to allow a fast growth with clockwise climbing. When flowers are harvested early in Autumn, and dependant of the hop variety, the composition and relative richness of hop resins is determined following a traditional classification in two main groups: aroma and bittering. A third one, dual-purpose (meaning high -acids content and valuable aroma properties) tends to become generally accepted mainly for commercial reason [12-14]. They rely on the tastes of hop flower teas and of the beer due to hop addition (either as dried cones, pellets or extracts) in the brewing process. This qualitative scoring is completed by quantitative biochemical analyses of a selection of hop secondary metabolites present in the thoroughly dried cones [15, 16].

In the last thirty years, several studies on genetic diversity among cultivated hop varieties and wild type hops have been published and used inbreeding programs continuously generating numerous new registered varieties. In 2016, the production of 249 commercial cultivars, adapted for various pedoclimatic conditions and bred mostly to fit with market driven product requirements, was reported by the International Hop Growers’ Convention (IHGC) [17]. Hopis produced in 30 countries with a total acreage for 2017 of about 55,000 ha. Germany and the USA, the two first producers, represented 2/3rd of the world production. Besides historical cultivation areas of the Northern hemisphere, the development of a hop business in Italy is also going on [18-20] and the Southern hemisphere contribution to the world hop production rely up to now on Argentina, Australia, New-Zeeland and South Africa [17]. Based on their knowledge accumulated over generations, hop breeders take advantages from the availability of fine biochemical analyses and of molecular biology technologies. Genetic distances based on various DNA polymorphisms, expressed sequence tags (ESTs) and other techniques generating vast number of data, allow the clustering of all available and future cultivars along with individuals from wild hop germplasm growing collections [21-26]. One of the very first published study showed that rDNA restriction fragment length polymorphism (RFLP) was able to segregate hops according to their geographical origins [27]. More recently, genomic and plastidial DNA tags identified accurately the continental origins of hops allowing new breeding program strategies [25, 28]. A still current aim of breeding programs is to combine in one variety both aromatic and bitter properties, with high yield, disease resistance and easy crop. Such hunted dual-purpose varieties can preserve aromatic properties without reducing the -acid contents and the targeted beer quality. Strong efforts have been engaged to find the suitable father plantable to bring bittering, respectively aroma, components in an aroma, respectively bitter, mother variety without altering it first qualification and so to generate suitable F1 plant populations expected to contain future and competitive new variety candidates. Searches for useful quantitative trait loci (QTL) have shown to be highly complex as described in the remarkable work of McAdam et al. published in 2013 [29]. In this reference, unfortunately, no mention about plastid or mitochondrial genome contributions can be read. With respect to cell energy (ATP production) and metabolism, the fundamental role of mitochondria is recognised [30-33]. The angiosperm mitochondrial genome size and organization differ considerably from one taxa to another and ranges from around 200 to at least 2,500 kbp [34]. The available hop mtDNA assembled sequence of 336,811 bp [2] is positioned at the lower size range. Although maternal inheritance in plants is predominant, biparental mitochondrial transmission have been reported in various taxa [35, 36]. The data published by Clark et al. [8] and the progress in mtDNA sequencing [2] encourage to overcome possible difficulties due to both size and mode of parental transmission to progeny of mitochondrial genome and to dynamize its study in hops which has sofar been timorous.

Here, within the 14,472 bpmitochondrial sequence of the Strisselspalt hop variety, a 560 bpregion was selected at the 5’ end of the gene encoding the  subunit of ATP synthase (EC 3.6.1.14), highly conserved among plants [37-39]. It contains a single nucleotide polymorphism acting as a signature that splits in two groups the 31 selected plants used in a still running hop breeding program and raises the question of maternally inherited mitochondrial genome in hops.