Diploid potatoes can be converted from an outbreeding species, in which self-pollination is prevented by a gametophytic self-incompatibility system, into one where self-pollination is possible, either through a dominant self-incompatibility inhibitor gene (Sli) or knockout mutations in the incompatibility locus. As a result, diploid F1 hybrid breeding can be used to produce genetically uniform potato cultivars for propagation from true potato seeds by crossing two near-homozygous inbred lines, derived from a number of generations of self-pollination despite inbreeding depression. Molecular markers can be used to detect and remove deleterious recessive mutations of large effect, including those in tight repulsion linkage. Improvements to the inbred lines can be made by introducing and stacking genes and chromosome segments of large desirable effect from wild relatives by backcrossing. Improvements in quantitative traits require a number of cycles of inbreeding and crossbreeding. Seed production can be achieved by hand pollinations. F1 hybrid planting material can be delivered to farmers as true seeds or young plants, and mini-tubers derived from true seeds.This paper reviews the progress and the way ahead in diploid F Solanum tuberosum) is the world’s fourth most important food crop after maize, rice, and wheat, with 359 million tonnes fresh weight (FW) of tubers produced in 2020 from 16.5 million hectares of land, in 163 countries, resulting in a global average yield of 21.8 t ha−1 . Most cultivated potatoes are tetraploids, and their ancestry can be traced back to autotetraploids (2n = 4x = 48) of the diploid cultigen S. tuberosum Stenotomum Group (2n = 2x = 24), which was domesticated from diploid wild species in the highlands of Southern Peru [1 hybrid breeding in 2008 [1 hybrid cultivars. This would allow potato breeders to reduce the genetic load of undesirable alleles through inbreeding and then combine desirable traits through hybridization, as also pointed out by Bachem et al. [The potato , chosen by Mendel for his experiments in plant hybridization that led to his laws of inheritance [Triticum aestivum) and rice (Oryza sativa) are inbreeding, seed-propagated crops; maize (Zea mays) is an outbreeding, seed-propagated crop; and the potato (S. tuberosum) is an outbreeding, but primarily clonally propagated, tuber crop. It can be argued that, as a consequence, maize breeding has benefited the most, and potato breeding the least, from underpinning genetics research [1 hybrid cultivars of outbreeding crops from genetically variable, open-pollinated ones. Thus, let us return to the breeding of diploid potatoes, starting with diploid potato germplasm.Darwin studied the effects of cross- and self-fertilization in 57 species of flowering plants from 52 genera and 30 families . He conderitance . Mendel’eritance . In retrresearch . Darwin research ,14. Fishresearch . FurtherS. tuberosum [Andean farmers still cultivate diploid landraces and named cultivars of the Stenotomum Group, the Goniocalyx Subgroup, and the more widely grown Phureja Group, of uberosum . Such geuberosum ,18,19. Fuberosum ,21,22. Duberosum .S. tuberosum to the diploid level through crosses with 2x Phureja Group ‘pollinators’ that induce dihaploid production by a form of parthenogenesis [S) locus was mapped to chromosome 1 by Gebhardt et al. [Since the 1960s, it has also been possible to reduce tetraploid ogenesis ,25. Subsogenesis ,27, althogenesis . Busse eogenesis  have desogenesis ,30, and ogenesis . Most diogenesis ,32, whict et al.  and Kauft et al. .1 hybrid breeding, but, as in all breeding programs, it will be important to use the best germplasm available for a particular target environment and particular end use. Breeding objectives will no doubt be expressed in terms of improving yield potential and tolerance to important abiotic stresses, and improving quality traits and resistance to important pests and diseases.In summary, there is no shortage of diploid germplasm available for diploid FBreeding diploid potatoes for clonal propagation can be conducted through the testing, selection, and vegetative multiplication of desirable clones from population improvement schemes, such as mass or family selection ,20,21,35Thus, in the selected genotype, as in the parents, deleterious recessive alleles are accommodated without adverse effects through heterozygosity.1 hybrid cultivars for propagation through TPS, one needs to produce homozygous inbred lines so that the cross between a pair of lines is genetically uniform. For each of the 12 pairs of chromosomes, the female parent will have two copies of the same chromosome, and the male parent two copies of a different chromosome  the ability to produce seed by self-pollination; (2) the ability to produce homozygous inbred lines with acceptable vigor and fertility, or sufficiently homozygous inbred lines to produce an F1 hybrid of acceptable phenotypic uniformity; (3) the ability to produce sufficient inbred lines for combinations to be found that are superior to existing cultivars, and to achieve this over cycles of inbreeding and crossbreeding for continued progress; and (4) the ability to produce large quantities of F1 seed for growing the hybrids commercially.The prerequisites for successful FSli) found in S. chacoense by Hanneman [S locus on chromosome 1 [Sli) was produced from self-compatible (SC) S. chacoense by seven generations of self-pollination, and found to be vigorous and fertile, as well as morphologically indistinguishable from S. chacoense plants that had not been inbred. It possesses a number of desirable agronomic traits, processing quality and resistance to Pectobacterium carotovorum soft rot and Verticillium dahlia wilt, as well as undesirable wild species traits, including unacceptably high levels of glycoalkaloids. Interestingly, it produced  tubers in a 14 h photoperiod, unlike many other wild species. Genomic information on M6 was provided when Leisner et al. [S. tuberosum Phureja Group clone DM1-3 516 R44 (DM1-3), which was used to provide the first published sequence of the potato genome [1 to produce an F2 population for quantitative trait locus (QTL) analysis. Kaiser et al. [Leptinotarsa decemlineata) in a diploid S. chacoense recombinant inbred line population derived from 308 F2 individuals after crossing M6 (self-compatible) and resistant accession USDA8380-1 . Fifty-five F5 families were analyzed, and all individuals contained at least one copy of the Sli gene; however, this was not sufficient for selfed fruit and seed production. Loci on chromosomes 3, 5, 6, and 12 were identified as novel targets for self-fertility improvement, and a major QTL for foliar leptine glycoalkaloid biosynthesis and Colorado potato beetle resistance was mapped to chromosome 2. Self-compatibility and resistance to Colorado potato beetle were introgressed into diploid breeding material with desirable tuber traits. Thus, germplasm useful to practical breeders is emerging from such genetics research.Successful self-pollination of diploid potatoes was first made possible by the use of the dominant self-incompatibility inhibitor gene  is in fact widespread in the cultivated gene pool of the potato plant, including the tetraploid cultivars Garnet Chili, Irish Cobbler, Early Rose, Kennebec, Russet Norkotah, Sierra Gold, Yukon Gold, Snowden, Atlantic and Mountain Rose; with Garnet Chili, Irish Cobbler, and Early Rose probably, and Russet Norkotah definitely, possessing two copies of the Sli allele. They concluded that cultivar Rough Purple Chili, introduced into the USA in 1851 by Goodrich [Sli KASP™ markers, Kaiser et al. [Sli to SC in the Michigan State University diploid germplasm, which represents diverse clones derived from multiple North American breeding programs. Although the Sli markers predicted SC in some germplasm, there were discrepancies, which emphasized the need to identify other genomic regions critical to SC and the role of the environment in the expression of genes involved in the SC reaction. For example, although M6 was confirmed as being homozygous for the SC Sli genotype at all six loci, the self-compatible clones 1S1 and DMRH-89, and a plant of S. chacoense accession PI 133664–40, were homozygous for the SI Sli genotype at the six loci.Clot et al.  found thGoodrich , is the Goodrich  found thGoodrich  also devr et al.  assessedSli gene, and that it is capable of interacting with multiple allelic variants of the pistil-specific S-ribonucleases (S-RNases), and thus functions as a general S-RNase inhibitor to impart self-compatibility to RH and other self-incompatible potatoes. RH has a pedigree derived from dihaploidized tetraploid commercial cultivars.Ma et al.  have conS-RNase gene (at S locus) of the diploid self-incompatible S. tuberosum Phureja Group clone S15-65, using the CRISPR–Cas9 system. The growth vigor and plant morphology of the mutant lines did not differ from those of clone S15-65, indicating that they could be used directly for breeding. The percentage of plants in the selfed families without the Cas9 cassette varied from 3.6% to 24.5%, thus demonstrating that self-compatibility can be achieved without introducing any exogenous DNA. Using the same method, the researchers also obtained S-RNase mutants in two more S. tuberosum Phureja Group clones (S15-47 and S15-76) and two S. tuberosum Stenotomum Group clones (S15-48 and S15-107). Hence, improved diploid clones from a population improvement scheme could be made self-fertile for the purpose of inbreeding and crossbreeding to produce F1 hybrid cultivars. Enciso-Rodriguez et al. [S-RNase gene. They achieved nine knockout lines (deletions/insertions) which transmitted self-compatibility to their progeny.Ye et al.  created z et al.  also useDiploid hybrid breeding occurred much earlier in other outbreeding crops. The six examples given in my book on plant breeding  fell intZea mays) naturally reproduces by wind cross-pollination, primarily, but not exclusively, as a result of having separate male and female flowers on the same plant (monoecious). However, it is also easy to self-pollinate, and hence proved suitable for both genetics and plant breeding research. The breeding research on the effects of inbreeding and crossbreeding in maize by East [1) hybrids from the 1960s [Allium cepa) are predominantly a cross-fertilizing, insect-pollinated, diploid species, although self-pollination occurs when the inflorescences of breeding material are simply bagged. F1 hybrids started to appear in the USA from the early 1950s, and in the Netherlands from the late 1960s [Daucus carota) are an outcrossing, insect-pollinated, diploid species with hermaphrodite flowers that are usually protandrous, although self-pollination is not restricted by incompatibility. Carrot hybrids started to become available in the 1970s and, since the 1980s, have been replacing open-pollinated cultivars worldwide [Maize , such as Brussels sprouts, cabbage, calabrese/broccoli, and cauliflower. Self-pollination is normally prevented by a sporophytic self-incompatibility system, as was first demonstrated and explored by Thompson [B. oleracea var. acephala) breeding, which started at the former Plant Breeding Institute in Cambridge in 1951 [Since the 1960s, F in 1951 . Inbred Secale cereale) is a diploid, cross-pollinated cereal with an effective gametophytic self-incompatibility system [Beta vulgaris) is another outbreeding crop in which self-fertilization is normally prevented by a gametophytic self-incompatibility system. Again, self-fertility was provided by a dominant gene (fS) [x CMS mm F1 × 2x N MM), where CMS is cytoplasmic-genetic male sterility, N is normal cytoplasm, and mm is the genotype required for the monogerm seed [Rye  . In receerm seed .In conclusion, in the three crops with a gametophytic self-incompatibility system, potato, rye, and sugar beet, reliable and successful self-pollination required the discovery and use of a dominant self-fertility gene.S. chacoense background, and eight self-incompatible (SI) ones, with introgressed disease and pest resistance traits of interest, were used to produce 50 offspring, from each of 24 SC × SI crosses, as the foundation population. Once underway, selected diploid germplasm was grown during winter in a greenhouse (16 h light/8 h dark photoperiod at 20–25 °C), checked for ploidy level , and screened for self-compatibility. The pollen from self-compatible diploid clones was bulked and used to pollinate the selected female plants (SC and SI). Seed extraction and germination took place in spring, and seedlings were transplanted to the field in summer. Field selections based on maturity and tuber-related traits took place in autumn. The cycle was repeated annually over a five-year period. Self-fertility increased from 16% to 86% of the population, male sterility decreased from 32% to 0%, and non-flowering decreased from 19% to 3%. Single-nucleotide polymorphism (SNP) markers (4885) revealed a slight decrease in the frequency of heterozygous SNPs (34% to 30%) and a reduction in linkage disequilibrium; despite this, genetic diversity was maintained. There was an increase in earliness for vine maturity, as well as an increase in tuber yield from 430 g to 522 g over four cycles, which is a respectable 5% increase per year. The improved population can be used as the starting point for an inbreeding and crossbreeding program leading to F1 hybrid cultivars. However, it might first be desirable to select for increases in the frequencies of major genes for disease and pest resistance, as well as other traits, in order to increase the frequency of desirable homozygotes in the population. The question thus arises, how easy is it to produce homozygous inbred lines of potatoes?In the USA, at Michigan State University, Alsahlany et al.  have sho1 hybrid potato breeding for TPS is to achieve the genetic uniformity not possible in tetraploid potatoes with the methods available, as reviewed by Golmirzaie et al. [One of the main reasons for conducting diploid Fe et al. , Ortiz [e et al. , Simmonde et al. , and Gope et al. . Hence, nH) at a given locus after a given number of generations (n) of selfing, as follows:0H is the proportion at the start of the selfing program. For example, starting with 1 for the F1 generation, by F6 (five generations) we have 1/32, or 3.125%, and by F9 (eight generations) we have 1/256, or 0.39%. The prediction assumes equal viability and fertility of the three genotypes, i.e., equal contributions to the next generation. However, if the heterozygote makes a greater contribution than the two homozygotes, which make lesser but equal contributions , it can be shown that the proportion of homozygotes at equilibrium is 1/(2s). Hence, when s is greater than ½, complete homozygosity never results, and, when s = 1, the proportion of homozygotes produced is ½ [s = 1 is when there is a very tight repulsion linkage involving two recessive lethal alleles (a and b) so that homozygotes Ab/Ab and aB/aB prove lethal, and only the heterozygote Ab/aB survives to reproduce.It was Mendel  who realced is ½ ,75. An ePopulation Genetics how this proportion can be calculated without knowing the number of loci through Fisher’s “theory of junctions” [v) of the plant species and its total map length (L), defined as half the number of chiasmata per nucleus, and measured in Morgans, together with an understanding of meiosis. It is assumed that, at the beginning, every locus is heterozygous. Then, after n generations, the proportion of lines homozygous at all loci is -me, where m is the number of heterozygous tracts (stretches of chromosome that are not homozygous) averaged over lines, provided n is large enough for m to be small , and the results are shown in For diploid potatoes, the respective values of However, Gale  pointed q = (μ/s)½ for a random mating population, where q is the equilibrium frequency of recessive allele a, μ is the frequency of mutations from A to a, and the relative fitness of aa is 1 − s, compared with 1 for AA and Aa. As μ is generally much smaller than s, it follows that q will be small and q2, the frequency of aa, will be very small.Textbooks on population genetics, such as that by Mather , give thGenetics of the Evolutionary Process, in terms of lethal, semi-lethal, sub-vital, normal, and super-vital chromosomes of Drosophila species. When the corresponding homozygotes are formed through inbreeding, a marked reduction in fitness, known as inbreeding depression, occurs. Hence, inbreeding depression is a very widespread phenomenon in outbreeding species. In contrast, in inbreeding species, homozygous recessives appear much more frequently than in a random mating population, and, if harmful, will tend to be eliminated by natural selection so that little inbreeding depression is found.In outbred populations, heterozygotes for the mutant will greatly outnumber homozygotes. However, although harmful recessive alleles at any given locus will be rare, individuals completely free of harmful recessive alleles at every locus on the chromosome (and hence in the whole genome) will also be very unusual in outbreeding species. Experimental results were summarized and discussed by Dobzhansky  in his 11 hybrid potatoes, Lindhout et al. [2 populations derived from three diploid clones , they identified 15 genomic regions with severe segregation distortions due to gametic (4) and zygotic (11) selection. Five regions contained recessive lethal mutations, and four regions contained mutations affecting growth vigor. In addition to the alleles with substantial deleterious effects, there were numerous mutations that had minor effects on fitness.It has also been argued that inbreeding depression is expected to be more severe in clonally propagated plants than in seed plants, and increases the difficulties in developing inbred lines. This is because a large number of deleterious mutations can accumulate over prolonged clonal propagation, although reproduction in potatoes in the Andes is probably a complex mixed sexual/clonal system; young plants in a field are a mixture of planted clones and recombinant genotypes from sexual reproduction . Lian ett et al.  did not t et al.  exploredSli gene donor (S. chacoense), followed by self-pollination for five generations (S5) (the detailed pedigrees are given in their paper). Serious loss of fertility was encountered, with only one of seven cross combinations reaching S5. Having started with 5040 plants from the seven families, just 43 out of 427 S5 plants produced selfed seeds. Furthermore, for RFLP (restriction fragment length polymorphism) and AFLP (amplified fragment length polymorphism) markers, the average percentage reduction in heterozygosity per generation (38.5%) was lower than the theoretically expected value (50%), indicating some resistance to homozygosity. None of the loci or chromosome sections were exclusively heterozygous in all of the advanced self-progeny.As diploid potatoes are outbreeders, inbreeding depression and resistance to complete homozygosity at all loci is expected over the generations of self-pollination. Phumichai et al.  demonstrS. chacoense carrying the Sli gene. The hybrids were extremely vigorous, and about half of them produced many berries upon self-pollination. However, the F2 plants showed weak growth, and many died in the field. Nevertheless, 10% of the surviving F2 plants proved self-compatible, and were used to produce the F3 generation. Undesired characteristics from the S. chacoense parent  were removed in a few generations. However, the first inbred lines, containing over 95% homozygous loci, produced few progenies, and the seedlings were extremely weak. Hence, new crosses were made between F3 and F5 inbred lines, followed by more intense selection over the subsequent selfed generations, and resulted in inbred lines with improved performance. By 2020, sufficient progress had been made for a single F9 line, named Solyntus, to be sequenced as the new standard reference in potato genome studies [S. tuberosum Phureja Group [S. tuberosum that is highly homozygous, vigorous, and self-compatible. Its estimated genome size is 710 Mb, with 0.3% of the genome estimated to be heterozygous and 89.7% of the genome unique. However, when an SNP (single nucleotide polymorphism) rate of 200 SNPs per 30 kb was taken as the threshold for heterozygosity, 4379 out of 21,776 windows showed a signal above this threshold, which is equivalent to 20.1% of the genome still being heterozygous, and much higher than expected for an F9 inbred line. The authors attributed this to a combination of unnoticed and undesired outcrossing during the generation of Solyntus, and a preferential selection of heterozygotes in the selfing generations due to inbreeding depression.More extensive results have come from the Solynta breeding and research program . They st studies . This seja Group . Solyntuws1), abnormal rooting 1 (ar1), lethal allele 2 (la2)), or growth vigor (plant architecture 1 (pa1), plant architecture 2 (pa2), and weak vigor 1 (wv1)). Generally, large-effect deleterious mutations were relatively dispersed throughout the genome, and hence could relatively easily be removed by selection. However, two recessive detrimental alleles, ws1 and pa1, were tightly linked in repulsion on the short arm of chromosome 1, and required larger populations and further analyses to find the desirable, but rare, recombinants.Further insight into the task facing potato breeders comes from the haplotype-resolved genome analyses of the self-compatible heterozygous diploid potato RH89-039-16 (RH) by Zhou et al. . RH has 1 hybrids through genome design, which is far more precise and effective than phenotypic selection. The method involves four steps: (1) selection or creation, as starting materials, of self-compatible diploid clones with low genomic heterozygosity, (2) analysis of the S1 population of this starting material to identify the segregation distortion regions (SDs) and genetic loci carrying large-effect deleterious mutations or controlling agronomic traits, (3) production of highly homozygous inbred lines by continuous selfing and genome-assisted selection, to purge deleterious mutations and stack beneficial alleles, and (4) crossing the inbred lines, derived from different lineages, to produce F1 hybrids for evaluation. Using this method, they were able to select two (PG6359 and E86-69) out of four clones for a starting material that had lower heterozygosity and fewer deleterious mutations. They were able to detect segregation distortion (SD) in the selfed families (S1) of the four clones at 3, 2, 6, and 5 loci, respectively. The progeny of the latter two clones became very weak, and seldom flowered after two generations of selfing. Starting at S1, based on genetic analyses, they eliminated deleterious or undesirable alleles as well as stacking four beneficial alleles for self-compatibility, normal leaves, fertility, and yellow flesh. From the first heterozygous self-compatible clone PG6359, they were able to achieve average genome-wide homozygosity of 48.32% (range 19.30% to 74.76%) in S1, rising to 97.54% (range 91.79% to 99.94%) in S5. They were also able to use genomic analysis and selection to detect recombinants among phenotypically green plants that broke the close linkage of two deleterious recessive mutations in repulsion phase at the end of chromosome 12, where led1 is a large-effect deleterious mutation and yl1 is a deleterious mutation for chlorotic yellow leaves rather than normal green ones  were also produced from clone E86-69. Crosses were then made between the two sets of inbred lines. The F1 hybrids between homozygous inbred lines were very uniform, whereas those derived from heterozygous parental lines showed a lot of segregation for tuber traits. Furthermore, the inbred-line-based F1 hybrids showed strong heterosis in growth vigor and yield in the greenhouse, with the yield of the best F1 hybrid combination 3.38 times that of the mid-parent value. Mini-tubers from true seed plants were used to assess the hybrids in the field in Yunnan Province, China (24°43′ N 98°58′ E). Due to severe inbreeding depression, the inbred lines performed very weakly in the field, and only a few tubers could be harvested. This was despite the elimination of deleterious mutations of large effect from the inbred lines, thus indicating the presence of many homozygous deleterious mutations of minor or medium effects. In contrast, the F1 plants grew well, and the average yield was twice that obtained in the greenhouse, thus indicating that the inbred lines complemented each other for the deleterious genes. The estimated tuber yields of the hybrids was around 40 t ha−1, close to the yield of Lishu 6 (45 t ha−1), the major tetraploid cultivar grown in the region, with 65% coverage of the local cultivated area. Furthermore, due to the self-compatibility and recovered fertility, the F1 hybrids produced abundant fruits. The reader is referred to the paper for more details [Inbred lines of similar homozygosity  in the yields of the parental inbred lines available from 1930 to 1980, and a corresponding 74% increase (5.27 t ha−1 to 9.16 t ha−1) in the yields of their hybrids [1 hybrid potatoes. Troyer [The comparison usually made for Fbreeding ,90. A febreeding  reportedbreeding  concludebreeding . Moreovebreeding . Experim hybrids ,95. In o. Troyer  attributB. napus, along with oilseed and forage rape, and provide an interesting contrast to the B. oleracea vegetables, such as Brussels sprouts (B. oleracea var. gemmifera). Brassica napus  is the allotetraploid of B. oleracea  and B. rapa  [S-alleles [B. napus is normally self-compatible, and swedes (B. napus var. napobrassica) can be treated as an inbreeding species that is tolerant to inbreeding, presumably because the ‘a’ and ‘c’ genomes complement one another.The Brassica vegetables provide an example of the difference between outbreeding and inbreeding species. Swedes are a vegetable and fodder crop that belong to the species pestris) , two out-alleles ,97,98,991 hybrid Brussels sprouts. The first F1 hybrid to be marketed from the program was cultivar Avoncross, with a marketable  yield 19% greater than its nearest competitor, cultivar Sherardian. The method developed for the field production of F1 hybrid seed was based on the self-incompatibility system; however, today, in the B. oleracea vegetables, cytoplasmic male sterility (CMS) is preferred, as briefly reviewed by Dixon [1 hybrids from sibs of the inbred parents [1 hybrid cultivar Gower for both marketable yield and sprout quality. The researchers concluded that in the short-to-medium term, inbred lines were uncompetitive compared with F1 hybrids.As early as 1966, Johnston , at the by Dixon  and Brad parents . Out of 6 families produced by SSD in a modest-sized breeding program in Scotland. Heterosis for yield does exist in swedes [1 hybrid cultivars produced from self-incompatible inbred lines [In contrast, in swedes, Bradshaw et al.  had no dn swedes , and caned lines , as proped lines ,107,108.ed lines . NeverthMy conclusion from this section is that diploid potatoes, like Brussels sprouts (and maize), are likely to remain an outbreeding crop that can be self-pollinated rather than become an inbreeding crop, such as swedes. The reason for this is that it is difficult with an outbreeding crop to avoid producing inbred lines that are homozygous for (recessive) deleterious genes at certain loci, whereas it is much easier to produce hybrids that combine different chromosomes that complement one another at these loci.B. napus) and autotetraploid potatoes (S. tuberosum). The gametophytic self-incompatibility system breaks down in autotetraploids, at least when two different alleles are present in the pollen [Finally, it is worth pointing out the contrast between allotetraploid swedes  alleles of large effect into new F1 hybrid cultivars, and accumulating desirable QTL alleles of small effect for improvements in quantitative traits where the number of loci is a key factor. We are now emphasizing positive selection for as many desirable (dominant) alleles of small effect as possible, rather than structural removal of many unfavourable (recessive) alleles of small effect [We have considered the elimination of deleterious mutations of large effect during the selfing generations leading to inbred lines, and have seen the value of using molecular markers to aid this process. Now we assume that diploid Fl effect ; nonethe1R) and W2(2R) are two (heterozygous) wild species with the two resistance genes, 1R and 2R, which may be homozygous or heterozygous. T1 and T2 are two elite diploid Tuberosum (homozygous) inbred lines. Selection in the F1, BC1, and BC2 generations is for the R-genes and, in the BC1 and BC2 generations, for as much of the Tuberosum genome as possible; ideally with no wild species genomes being left in the BC2 generation, apart from the R-genes. Finally, selfing the selected BC2 plants produces homozygotes for the R-genes  from potato wild relatives and landraces into elite lines as they emerge. Today, the introgressions can be performed quickly and efficiently through molecular-marker-assisted backcrossing, in which there is precise selection for the desired products of meiosis in each backcross generation ,116,117. R-genes .R-genes for resistance to Phytophthora infestans, the cause of late blight, namely Rpi-tar1 from S. tarijense [vnt1 from S. venturii [1W1/T2T2/T3T3…, T1T1/W2W2/T3T3…, T1T1/T2T2/W3W3…T118T118/T119T119/W120W120). In practice, segments will be overlapping and of variable length; despite this, the effect of each genetically fixed segment on traits of interest, relative to the elite line, can be determined using appropriate trials to see if any of the substitution lines are superior to the elite line. Hence, using these backcrossing methods allows the fast, targeted, and predictable breeding of new potato cultivars.Su et al.  have shoarijense  and Rpi-venturii ,121. Theventurii , with onventurii . In an i1 hybrid cultivar for TPS, is not that different to the introgression and combining of genes in a tetraploid cultivar for clonal propagation, once one thinks in terms of cycles of crossing and selection [Although alleles and chromosome segments of large effect can be stacked into an elite inbred line, if further improvements in commercially important traits require crossing to other elite lines, the genes of large effect will segregate, and combining more than, for example, eight of them into a new cultivar will require careful planning over a number of generations. Indeed, I would suggest that the introgression of genes, and combining them in a diploid Felection . There welection . It is n1) hybrids as the frequencies of the desirable alleles increase as a result of selection. Starting with an additive-dominance model for a single locus, where p and q are frequencies of alleles A and a, we have the following:1 hybridsGenotypes of FAAAaaaGenotype frequencyp2pq2q2A in F1 hybridsFrequency of 1½01 hybrid meansFada−Genotypes of inbred linesAAAAaaaaGenotype frequencyp2pqpqq2A in inbred linesFrequency of 1100Inbred line meansaaa−a−The theory and practice of diploid inbreeding and crossbreeding can be found in books on plant breeding and genetics ,124, andm is an arbitrary mean for all of the inbred lines when p = q = ½, EV is the plot-to-plot environmental variation in the assessment trials of hybrids and inbred lines, and r is the number of replicates in the trials:Then, summing over loci (Ʃ), we have the following, where 1 hybrid meansMean of F1 hybrid meansVariance of FMean of inbred line meansVariance of inbred line meansR) can be determined from the following general formula [Responses to selection , S is the selection differential (mean of selected group minus mean of unselected population), and dM/dp is the rate of change in the population mean with the change in allele ‘A’ frequency.Here, Thus, the response to selecting the best inbred lines, seen in the next population of inbred lines produced by crossing the selected inbred lines in all combinations and then producing a new set of inbred lines from the crosses without any further selection, is as follows:1 hybrids rather than in the next population of inbred lines cannot be determined so simply, as one needs to take into account the difference in population means.In other words, the selection differential is multiplied by the heritability of the inbred line means, which can be determined from the assessment of the inbred lines. The predicted response to selection will give an indication of likely progress in practice. In contrast, the response in the population of F1 hybrids before starting the next cycle of inbreeding, but it is probably more useful to consider the following simple example of the effect of changes in favourable allele frequencies on the inbred lines and their F1 hybrids.We could also consider selecting among the Fa = d = 1) at each of 12 loci, one on each of the 12 pairs of chromosomes of diploid potatoes, we can produce the results shown in p = ½ is set at 12 for the inbred lines.If we assume complete dominance  determines the magnitude of the effects (heterosis and inbreeding depression). The means plus one standard deviation indicates that the best hybrid will be better than the best inbred line.As the frequency of the favourable allele at each locus increases , so does the mean and the mean plus one standard deviation for the inbred lines, and likewise for the hybrids. At a given allele frequency, one can consider the mean of the hybrids higher than the mean of the inbred lines as a result of heterosis, or the mean of the inbred lines lower than the mean of the hybrids as a result of inbreeding depression; the cause of both being the directional dominance for the higher yield. The degree of dominance  to 12 (yield 24) loci either heterozygous or homozygous for the advantageous allele. The inbred lines show inbreeding depression with a mean of 12 compared with 18, the mean of the F1 hybrids. The variance  of the inbred lines (12) is greater than the variance of the F1 hybrids (9), which has an additive (6) and dominance (3) component. However, the higher variance does not compensate for the lower mean, so that 39.1% of the F1 hybrids have a yield greater than or equal to 20 compared with only 1.9% for the inbred lines. Furthermore, the frequency of the best inbred line is 1 in 4096 compared with (approximately) 130 in 4096 for the frequency of the equivalent best single crosses. However, the best inbred line is only 3.46 standard deviations from the mean of the inbred lines, and the best single crosses are only 2.00 standard deviations from the mean of the single crosses. In other words, with only 12 loci segregating, the best genotypes is quickly achieved and a selection limit is reached .ariation . With 12n) affecting the trait increases, the relative frequency of the best single crosses over the best inbred line increases . With 96 loci, at p = ½, the best inbred line is now 9.80 standard deviations from the mean of the inbred lines, and the best single crosses are 5.66 standard deviations from the mean of the single crosses. Hence, a number of cycles of inbreeding and crossbreeding will be required to achieve the best genotypes, as the selection differential in any modest-sized breeding program is likely to be around 2.00 standard deviations, and the heritability is likely to be less than the maximum of 1.00 [As the number of segregating loci . It is an inbreeding glasshouse and field crop in which the older, open-pollinated landraces were largely replaced by hybrids during the second half of the 20th century [1 hybrid breeding in all of the outbreeding crops mentioned earlier from Bradshaw [Another crop where F century . The han century . Emascul century . It is a century  thought Bradshaw , requireBipolaris maydis (formerly Helminthosporium maydis), the cause of southern corn leaf blight [1 hybrids using CMS, one is seeking maximum heterosis (hybrid vigor) between maintainer and restorer inbred lines based on their combining abilities. Hybrid rye is also based on cytoplasmic male sterility, and a restorer as pollinator [Maize has separate female and male flowers so that hybrid seed can be produced in isolated crossing blocks (seed fields) in which the female parent is detasseled, and in which the most common planting pattern is one row of pollen parents to four rows of seed parents . Since 1f blight ,133. CMSf blight ; the impllinator .1 hybrid breeding, were the B. oleracea vegetables , onions, carrots, and sugar beet. Today, hybrid seed production for all of these crops uses CMS, but there is no need for a restorer as the hybrids are non-flowering vegetable or beet crops; in fact, premature bolting is highly undesirable. Nevertheless, the use of CMS in testcrosses, followed by assessment of male-sterility, divides the germplasm into maintainers and restorers. Onions provide a good early example where a source of CMS (CMS-S) still used today was first discovered in 1925, and whose genetics was worked out by Jones and Clarke [msms at a nuclear male-fertility-restoration locus (Ms), so that male-sterile plants have the composition S-msms. Male-sterile plants can be seed-propagated by crossing S-msms  with N-msms  and harvesting seed from the female parent. They can then be used as the female parent in hybrid production. The nuclear genotype of the maintainer line  can be very similar to that of the female parent as a result of repeated backcrossing during maintenance. A high frequency of the Ms allele in germplasm of particular interest to a breeder makes finding a maintainer more difficult. As an onion crop does not flower, the unrelated male inbred parent used in hybrid production can have genotype N-MSMS or N-msms , or even S-MSMS. Details of a hybrid onion breeding scheme can be found in the review by Shigyo and Kik [The other diploid outbreeding crops considered by Bradshaw , as examd Clarke . CMS-S r and Kik .1 hybrid potato production in the same way as just described for onions, and this prospect will probably stimulate more research into the topic. The types of cytoplasm found in potatoes and their wild relatives, and their associations with male sterility and nuclear restorer genes, have been briefly reviewed by Ortiz and Mihovilovich [S. stoloniferum and S. demissum, respectively, are commonly associated with male sterility, unlike the A and P cytoplasms in Andigena and Phureja Group potatoes, respectively. Attempts may also be made to exploit the segregation at the Sli locus for self-compatibility/incompatibility in diploid F1 hybrid potato production. In the meantime, the important point is that the use of hand pollination means that there are no constraints on the use of diploid inbred lines of potatoes as female or male parents. It is too early to say if heterotic groups will emerge in the diploid potato germplasm. A heterotic group  is the germplasm that, when crossed with the germplasm from another heterotic group , exhibits a higher degree of heterosis than when crossed within its own group; thus, 1 × 2 hybrids are superior to both 1 × 1 and 2 × 2 hybrids. Heterotic groups in maize were established empirically, and their origin is not as clear-cut as once thought, as can be seen in the results of van Heerwaarden et al. [Male sterility could be used in diploid Fvilovich  and by Bvilovich , and morvilovich . Seven dn et al. . Where hn et al. .1 hybrid potato breeding is not quite as obvious as one might first think. All of the crops considered in this paper, apart from potatoes, are (true) seed-propagated, where F1 hybrid cultivars replaced open-pollinated ones. The hybrids were genetically more uniform and higher yielding; it was easier to combine a number of desirable traits in a hybrid; and there were commercial incentives, as hybrids do not breed true, and hence their seed cannot be saved for propagation. Thus, in maize, as we have seen, open-pollinated cultivars were replaced by double-cross hybrids, which in turn were replaced by single-cross hybrids, and there were also improvements in maize agronomy [−1 [−1 by 1995, with about half the increases resulting from improved agronomy and half from the change to hybrids and improvements in those hybrids. In Germany, to take another example, by 2009, the best rye hybrids were outyielding the best open-pollinated cultivars by 15–20% [Assessing the progress and success in diploid Fagronomy ,137. Thenomy [−1 ,137. They 15–20% . Account1 hybrid potatoes would be the current cultivars being grown for propagation through TPS. However, when Chilver et al. [1 hybrids. Furthermore, they were considering the net benefits of replacing vegetative propagation of potatoes with TPS technologies. Now, the first assessments of diploid hybrids are also being made in comparison with tetraploid cultivars for tuber propagation. Three examples will suffice.The obvious comparisons for diploid Fr et al.  reviewed−1, with an overall mean of 31.4 t ha−1, and the tetraploid cultivars between 52 and 101 t ha−1, with an overall mean of 72.1 t ha−1. Hence, on average, the diploid fresh weight yield was 43.6% that of the tetraploids, mainly as a result of the lower weight of individual tubers (71 vs. 177 g on average). The diploids had a higher number of stems (25.6 vs. 14.9 stems m−2) but fewer tubers per stem (1.8 vs. 2.9 tubers), resulting in approximately equal numbers of tubers per unit area (45.2 vs. 43.5 tubers m−2). The first scientific paper from the Solynta breeding and research program on the contribution and stability of yield components of diploid hybrid potatoes came from crosses made in the winter of 2015/2016 between inbred parents that had been selfed for five to seven generations . Seedlin1 hybrid potato for the tropical highlands of East Africa, where clean planting material is a problem, and hence TPS is an attractive proposition. In 2016, they trialed 10 representatives (out of 203) of their first experimental hybrids (crosses between F4 lines made in winter 2014/15) in East Africa at 1680 masl. Seedlings were raised from botanical seeds, and six weeks later transplanted to the field into irrigated ridges. Tubers were harvested four months later when all of the plants had died. The diploid hybrids yielded between 8 and 29 t ha−1 compared with average yields of potatoes in East Africa of 5 to 14 t ha−1. The best hybrid had yielded 26 t ha−1 in a Netherlands trial the previous year. These are just preliminary results, but indicate the potential of diploid hybrids in East Africa and the yield to be made up on tetraploid cultivars in the Netherlands that yield 35 to 40 t ha−1. Lindhout et al. [Another early result from the Solynta team is also worth mentioning. De Vries et al.  reportedt et al.  have put−1, close to the yield of Lishu 6 (45 t ha−1), the major tetraploid cultivar grown in the region, with 65% coverage of the local cultivated area. Furthermore, due to the self-compatibility and recovered fertility, the F1 hybrids produced abundant fruits.As mentioned earlier, in Yunnan Province in China, Zhang et al.  estimate1 hybrids to be assessed were lower-yielding than clonally propagated tetraploid cultivars, Jansky and Spooner [1 hybrids, but more experimental results are required before reaching a conclusion. One indication will come from population improvement programs conducted over short cycle times, such as that in the USA at Michigan State University, where Alsahlany et al. [1 hybrids for TPS propagation, and the breeding of tetraploid cultivars for clonal propagation, both rely on additive genetic variation for continued progress in improving quantitative traits over cycles of crossing and selecting. In any given cycle, however, the best genotype selected, whether a hybrid or clone, exploits both the additive and non-additive genetic variation available in the cycle. Finally, both breeding methods can incorporate marker-assisted selection of major genes and QTL alleles of large effect, as well as genomic selection for polygenic traits, and both can incorporate gene editing and genetic transformation.Although the first diploid F Spooner  have argy et al.  achieved1 hybrid cultivar for TPS propagation, or a tetraploid cultivar for propagation from tubers? Hence, I will finish with some practical considerations.Further research is also required to determine whether or not TPS crops are intrinsically lower-yielding than crops grown from more nutrient-rich tubers. I would, however, suggest that the major issue facing potato breeders concerns the target environments for, and end users of, the cultivars that will emerge from their breeding programs. Which makes more practical sense in a given situation, a diploid F1 hybrid cultivars for TPS propagation and cultivars for clonal propagation lies in their maintenance and production. For both types of cultivars, there must be a definitive stock, against which further multiplications can be checked. Here, I want to concentrate on the biology; however, a breeder cannot ignore the legal requirements in any territory (country or group of countries) for the registration, multiplication, and protection of new cultivars, nor the political, social, and economic context of the work. Details can be found in my books on plant and potato breeding [1 hybrid potato breeding, and thus help to ensure food security.All potato breeding programs start with hybridizations that will either be cross-pollinations or self-pollinations. The difference between diploid Fbreeding ,35. Beumbreeding  have expbreeding  have arg1 hybrid cultivars are produced by crossing two inbred lines that can be maintained as true seeds by self-pollinations, and which commercial companies will want to keep private and secure to protect their investment [Diploid Fvestment . The cul1 hybrid cultivar, therefore, starts life as true potato seed from which plants are grown, whereas a clonally propagated cultivar starts life as fully rooted in vitro plantlets. The success of both types of cultivars will partly depend on how they are made available to farmers for planting, or, in other words, the cost and quality of the seed system used to provide the planting material.A diploid FLow-income countries are characterized by informal seed tuber systems with very low use of certified seed, often below 10% , where fIn contrast, high-income countries are much more likely to have a commercial seed tuber industry, cultivar protection, and harmonization of standards. A typical seed tuber production scheme is likely to start with fully rooted in vitro plantlets (transplants) from which mini-tubers are commercially produced in a greenhouse or screenhouse with high health status . The tra1 hybrid cultivars, breeders and seed producers already had experience producing planting material for farmers from TPS. The methods fell into two categories; those in which there was no pre-propagation before commercial production, and those in which a multiplication phase intervened [TPS cultivars and production systems will need to prove superior to those achieved with conventional cultivars through modern methods of rapid multiplication of ‘disease-free’ seed tubers, if they are to replace them. Before the advent of diploid Ftervened . The firtervened  showed ttervened . Hence, tervened . Furthertervened  also contervened  also con1 hybrid potatoes provide the means to grow genetically uniform cultivars from true potato seed (TPS). Diploid germplasm is available in the form of potato wild relatives, landraces, cultivars, and dihaploids from tetraploid cultivars. Reliable self-pollination of diploid potatoes is possible through the use of the dominant self-incompatibility inhibitor gene Sli, initially found in S. chacoense, but later also in the cultivated gene pool. Self-compatibility can also be achieved through knock-out mutations in the S-locus that normally controls gametophytic self-incompatibility. Diploid potatoes can, therefore, be converted from an outbreeding species, which cannot be self-pollinated, into one which can, but are unlikely to become an inbreeding crop.Diploid F1 hybrid breeding and production, despite inbreeding depression and resistance to complete homozygosity. Molecular markers can be used to detect and remove recessive deleterious mutations of large effect, including those in tight repulsion linkage, and also to stack beneficial alleles. Genes  and chromosome segments of large desirable effect can be introgressed from wild relatives and stacked into elite inbred lines by backcrossing, but will segregate when the elite lines are used in further cycles of inbreeding and crossbreeding. The number of cycles of inbreeding and crossbreeding required to produce the best possible inbred lines and F1 hybrids for quantitative traits increases with the number of segregating loci affecting the trait. Breeding schemes used for diploid F1 hybrid potatoes will depend on the method used for F1 hybrid seed production, and whether or not heterotic groups of germplasm are found, but all will require the inbred lines to be selected for vigor, fertility, and combining ability for quantitative traits. Currently, diploid F1 hybrids are lower-yielding than tetraploid clones as a result of the lower weight of individual tubers; however, the likely relative rates for future improvements are not clear, although both will depend on the additive genetic variation in the germplasm.Inbred lines of potatoes can be produced by a number of generations of self-pollination, with sufficient levels of homozygosity, vigor, and fertility, for F1 hybrids for TPS and tetraploid clones in any given situation, the economics and practicalities of producing clean planting material is a major consideration. Diploid F1 hybrids start from true seeds, whereas clones start from pathogen-free micro-plants. Both types of cultivars require a “seed system”, whether true seed or seed tubers, to deliver planting material to farmers. This can be true seeds, young plants, or mini-tubers for F1 hybrids, and young plants or mini-tubers for clones, with further limited field multiplications to meet demand, which should be fewer from TPS. As potato is a major food crop, breeding will continue to operate in a social, economic, political, and legal context in which food security is both a practical and moral issue. Seed production is currently achieved by hand pollinations, and hence poses no constraint on the use of inbred lines as female or male parents, but cytoplasmic male sterility could become the preferred option for cheap, large-scale seed production. In deciding between diploid F


D), which is used as fertilizer. Antimicrobials and ARGs can be transferred to agricultural land, which increases their concentrations in the environment. The concentrations of 13 antibiotics in digestate samples from biogas plants (BPs) were investigated in this study. The abundance of ARGs encoding resistance to beta-lactams, tetracyclines, sulfonamides, fluoroquinolones, macrolide-lincosamide-streptogramin antibiotics, and the integrase genes were determined in the analyzed samples. The presence of cadmium, lead, nickel, chromium, zinc, and mercury was also examined. Antimicrobials were not eliminated during anaerobic digestion. Their concentrations differed in digestates obtained from different substrates and in liquid and solid fractions (ranging from 62.8 ng/g clarithromycin in the solid fraction of sewage sludge digestate to 1555.9 ng/L doxycycline in the liquid fraction of cattle manure digestate). Digestates obtained from plant-based substrates were characterized by high concentrations of ARGs (ranging from 5.73 × 102 copies/gDcfxA to 2.98 × 109 copies/gDsul1). The samples also contained mercury (0.5 mg/kg dry mass (dm)) and zinc (830 mg/kg dm). The results confirmed that digestate is a reservoir of ARGs (5.73 × 102 to 8.89 × 1010 copies/gD) and heavy metals (HMs). In addition, high concentrations of integrase genes (105 to 107 copies/gD) in the samples indicate that mobile genetic elements may be involved in the spread of antibiotic resistance. The study suggested that the risk of soil contamination with antibiotics, HMs, and ARGs is high in farms where digestate is used as fertilizer.Antimicrobials and antibiotic resistance genes (ARGs) in substrates processed during anaerobic digestion in agricultural biogas plants (BPs) can reach the digestate ( Due to growing levels of waste generation around the world and a higher demand for alternative and environmentally friendly energy, anaerobic digestion (AD) is becoming an increasingly popular method of stabilizing various types of organic matter substrates . The popMost BPs use manure , sewage Most antimicrobials, ARB, ARGs, and HMs present in digestate originate from substrates that are used in the AD process. Sewage sludge, the byproduct of wastewater processing in wastewater treatment plants (WWTPs), is also used as feedstock in BPs, and it contains large quantities of drugs, ARB, ARGs, and HMs  because In digestate, pharmaceuticals and HMs can also originate from manure and agricultural wastes that are rich sources of mineral substances and microorganisms and are most widely used as substrates in AD. According to the literature, global annual antibiotic consumption in livestock production has been estimated at 172 mg/kg in pigs, 148 mg/kg in chickens, and 45 mg/kg in cattle . In ChinDrugs that are introduced to the environment with digestate-based fertilizers can be highly toxic, and they can cause selection pressure which increases the concentrations of ARGs in soil and contributes to antibiotic resistance (AR) . In addiThe rate at which chemotherapeutics and HMs are degraded in soil is determined by temperature, soil texture, organic carbon content, and the resistance profile of soil-dwelling microorganisms . PharmacAntibiotics and HMs present in the environment can contribute to the spread of ARGs and ARB in soil, and they can trigger the emergence of new ARGs or increase their concentrations in a given environment . AntimicblaTEM, blaOXA, cfxA), tetracyclines , sulfonamides , fluoroquinolones , and macrolide-lincosamide-streptogramin (MLS) class antibiotics (ermF). The prevalence of class 1 and 2 integrase genes (intI1 and intI2) was also determined in digestate samples. The environmental implications of uncontrolled transmission of drugs and ARGs to agricultural land were assessed.The digestate can be considered a rich source of valuable minerals, but at the same time, unfortunately, it can also be a reservoir of antibiotics, ARGs and heavy metals for plants and microorganisms. There is a large research gap in the literature related to this topic, because there are still only few data on the content of ARGs, HMs, and pharmaceuticals and their persistence in the fermentation mass based on various substrates, which during soil fertilization with digestate can significantly affect their content in soil and edible vegetables. So far, there are no studies available with the content of so many antibiotic groups, HMs, and ARGs in digestate based on different substrates and the possible environmental implications for the soil–plant–animal–human system related to their transfer to the soil along with digestate used as fertilizer. Therefore, in this preliminary study, digestate samples from agricultural BPs stocked with various substrates, including sewage sludge, animal manure, and green waste, were analyzed for the presence of beta-lactam , tetracycline , sulfonamide , fluoroquinolone , macrolide (clarithromycin), lincosamide (clindamycin) antibiotics, and dihydrofolate reductase inhibitors (trimethoprim). Digestate samples were also examined for the presence of HMs such as Cd, Pb, Ni, Cr, zinc (Zn), and Hg, as well as genes encoding resistance to beta-lactams : BP1S, BP1W, BP2S, BP2W, BP3S, and BP3W.The presence and concentration of drugs and HMs, as well as the presence and abundance of ARGs, were examined depending on the substrate composition of digestate samples. Digestates were sampled in winter and spring, which differ in the availability of various substrates and weather conditions (such as temperature) and are characterized by increased disease incidence and, consequently, antibiotic consumption. Digestate samples were collected directly from the outlets of full-scale bioreactors operating under mesophilic conditions. Digestate produced by all three BPs is used as organic fertilizer in agricultural production.The analytical procedure for identifying antibiotics in the preliminary study was based on the methods that had been used in our previous research ,27,28. DThe limit of quantification (LOQ) was set at 0.1 ng/L of fresh liquid digestate fraction and 0.1 ng/g of fresh solid digestate fraction.The presence and concentrations of six HMs, i.e., Cd, Pb, Ni, Cr, Zn, and Hg in digestate samples were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES). The analyses of digestate samples were conducted in an accredited laboratory of the Institute for Ecology of Industrial Areas (IETU), Katowice, Poland ,30.® . The concentration and quality of the isolated gDNA were evaluated with the Multiskan Sky spectrophotometer . Additionally, gDNA was isolated in triplicate from each sample, and the isolates were pooled to obtain a representative gDNA sample for each digestate sample. The samples were freeze-stored (−20 °C) until analysis.Digestate samples (2 g) were placed in 2 mL Eppendorf tubes and centrifuged at 8000 rpm for 10 min. The supernatant was discarded after centrifugation. Genomic DNA (gDNA) was isolated from the pellet in triplicate, according to the instructions provided by the manufacturer of the Fast DNA Spin Kit for SoilblaTEM, blaOXA, cfxA), tetracyclines , sulfonamides , fluoroquinolones , and macrolide-lincosamide-streptogramin (MLS) class antibiotics (ermF) were analyzed in digestate samples. The prevalence of class 1 and 2 integrase genes (intI1 and intI2) was also determined. Genes conferring resistance to the above classes of antibiotics were analyzed because these antimicrobials are widely used in agriculture and in human and veterinary medicine. The presence and abundance of integrase genes were examined to assess the degree to which MGEs participate in the spread of AR. Antibiotic resistance genes were quantified in the quantitative Polymerase Chain Reaction (qPCR) in the LightCycler® system . The SYBR Green dye  was used in the reaction mixture with a total volume of 15 µL. The qPCR mix contained 0.8 µL of the DNA matrix, 0.4 μL of the corresponding primer with a concentration of 10 μM, 7.5 μL of SYBR Green, and 5.9 μL of RNase-free water. Genes were amplified in triplicate, and amplification parameters are presented in ® v. software (version 1.5.0). The number of gene copies was expressed in terms of absolute abundance (AA) in 1 g of digestate (ARG copy number/gD).The presence and abundance of genes encoding resistance to beta-lactams  and HMs concentration such as Cd, Pb, Ni, Cr, Zn, and Hg. Moreover, non-parametric Spearman’s correlation analysis was performed to indicate the effect of antibiotic concentration on the ARGs and integrase genes studied. The results were regarded as statistically significant at p < 0.05. The results of statistical analyses were visualized with the use of GraphPad Prism 8.4 software (v. 9.5.0).Significant differences between ARG and HM concentrations in samples were determined in the Kruskal–Wallis non-parametric test (Statistica v. 13.3). The correlations between ARG abundance, antibiotic concentrations, and HM concentrations in samples were determined in the Principal Component Analysis (PCA). Principal Component Analysis was applied using as variables all data from samplings BP1S, BP1W, BP2S, BP2W, BP3S, and BP3W regarding the concentration of 13 antibiotics such as AMO, AMP, TC, OXY, DOX, CHLOR, SMX, SFD, ENF, CIP, CLR, CLD, and TRI; ARGs concentration, such as p < 0.05). None of the examined digestate samples contained SMX, OXY, CHLOR, AMO, or AMP.The study revealed that 8 out of the 13 tested antibiotics were present in digestate samples. The presence of pharmaceuticals was determined by the composition of the examined digestates. Digestate samples differed significantly in antibiotic concentrations   between liquid and solid fractions. In sample BP1W, TC concentration was determined at 1164.4 ng/g in the solid fraction and 368.7 ng/L in the liquid fraction. In sample BP1S, TC concentration was determined at 464.8 ng/g in the solid fraction and 275.6 ng/L in the liquid fraction.The analyzed digestate samples contained TC and DOX antibiotics from the tetracycline class. Tetracycline was identified in samples BP1W and BP1S, in both liquid and solid fractions of the examined matrix. However, TC concentrations differed significantly . Doxycycline was identified in the samples collected from two out of the three analyzed BPs . Samples BP1W and BP1S were obtained from a BP processing sewage sludge, whereas samples BP2W and BP2S were collected in a bioreactor stocked with cattle manure and maize silage. Doxycycline was identified in nearly all of the above samples, in both solid and liquid fractions. Only the liquid fraction of sample BP1W was free of DOX. In the solid fractions of samples BP1W, BP1S, BP2W and BP2S, DOX concentration ranged from 218.1 ng/g to even 1282.5 ng/g. In the liquid fractions of samples BP1S, BP2W and BP2S, DOX concentration was determined in the range of 620.4 ng/L to 1555.9 ng/L. In the studied digestate samples, DOX was present in higher concentrations than the remaining antibiotics. Doxycycline levels were influenced by the substrate composition of digestate samples, but not by digestate fraction (solid or liquid).Significant differences in DOX concentrations were observed between digestate samples, depending on their substrate composition . Sulfonamides were identified in liquid fractions of samples BP1W, BP1S, and BP2W . Samples BP1W and BP1S were obtained from a BP processing sewage sludge, whereas the sample BP2W was collected from a bioreactor stocked with cattle manure. Sulfonamide concentrations ranged from 98.2 ng/L to 136.5 ng/L. According to other authors, SFD are effectively biodegraded during AD, but their removal rate can be affected by the conditions inside the bioreactor, such as the presence of iron, sulfates, and methane-producing microorganisms [2 and –CH3 electron donating groups in the drugs’ molecular structures. The biotransformation of SFD during AD can lead to the reduction in the electron-withdrawing sulfonyl group [Significant differences in the presence and concentrations of SFD were noted in solid and liquid fractions of digestate samples . This drug was not identified in the liquid fraction. Enrofloxacin was detected only in the solid fraction, and similarly to CIP, it was identified in samples BP1W and BP1S (digested sewage sludge) at concentrations of 147.1 and 387.9 ng/g, respectively .Ciprofloxacin was identified in samples BP1W and BP1S collected from a BP stocked with sewage sludge. Ciprofloxacin concentration was significantly higher in the solid fraction of sample BP1W (145.3 ng/g) than sample BP1S (92.4 ng/g) .In sewage sludge digestate (BP1W), CLR and CLD concentrations were significantly higher (more than three times) in the liquid fraction  than in the liquid fraction  of sewage sludge digestate (BP1S) . Trimethoprim contains two aromatic rings, a double aminated pyrimidine ring and phenyl-trimethyl ether. According to the literature, this drug is effectively biodegraded (in more than 80%) in both liquid and solid fractions of organic matter [2, –OH, –CH3 and –OCH3, in its molecular structure, which increases its susceptibility to biodegradation. Moreover, the drug has different biodegradation pathways under anaerobic conditions, depending on the availability of electron acceptors, both inorganic  and organic (such as sulfoxide and dimethyl sulfide) [Trimethoprim was identified in the solid fraction of samples BP1W and BP1S at concentrations of 66.8 ng/g and 226.1 ng/g, and in the solid fraction of sample BP2S (cattle manure and maize silage digestate) at 261.9 ng/g  . The presulfide) ,52,53, wThe concentrations of the analyzed antimicrobials were worryingly high in the studied samples, and they could induce irreversible changes in the environment by promoting the spread of AR and compromising the therapeutic efficacy of pharmaceuticals in human and veterinary medicine. According to the literature, predicted no-effect concentration (PNEC) values for resistance selection range from 8 ng/L to 64 μg/L , whereasThe composition of individual digestate samples has a significant impact on the availability and degradation of antibiotics. For example, sulfadiazine and ciprofloxacin are widely used antibiotics in both human and veterinary medicine, and their residues are among the most frequently detected pharmaceuticals in the environment . SulfadiFurthermore, according to many authors ,48, drug3 to 2.25 × 106 copies/g of digestate. The statistical analysis based on the Kruskal–Wallis non-parametric test did not reveal significant differences in ARG copy numbers between the analyzed samples  (data not shown). The abundance of ARGs was highest in sewage sludge digestate (samples BP1S and BP1W) and digestate derived from a mixture of cattle manure and maize silage (samples BP2S and BP2W).As demonstrated in D compared with the control [intI1 and intI2 play an important role in the transfer of ARGs during AD. The gene intI1 is the most ubiquitous integrase gene in the environment which, together with sulfonamide resistance genes, has recently been recognized as an indicator of contamination with ARB, ARGs, and other anthropogenic pollutants. Integrase genes are implicated in the HGT mechanism; therefore, integron-associated genes should be included in environmental AR analyses [According to Koniuszewska et al.,  antimicr control . An incr control . Accordi control ,16, intItetA and tetM genes encoding resistance to tetracyclines was high in the analyzed digestate samples. The abundance of the tetM gene ranged from 105 to 106 copies/gD, whereas the abundance of the tetA gene was determined at 104 copies/gD in most samples. The tetA gene was more prevalent only in sewage sludge digestate (from 105 copies/gD in sample BP1S to 106 copies/gD in sample BP1W).The abundance of tet genes are ubiquitous in both Gram-negative and Gram-positive bacteria, and most bacterial tet genes are associated with mobile plasmids, transposons, conjugative transposons, and integrons. These genes confer tetracycline resistance via an active efflux of tetracycline (tetA) and by preventing bacterial ribosomes from binding to the drug (tetM) [tet genes in the analyzed digestate samples. The high abundance of ARGs suggests that digestate-based fertilizers could pose a threat to human health. Moreover, digestate offers a supportive environment for the development of AR because it is characterized by high microbial diversity and a high content of organic carbon, nitrogen, phosphorus, and other nutrients that promote bacterial growth, even in the presence of multiple antibiotics [The g (tetM) ,55. The ibiotics .sul1 was the most abundant gene in all digestate samples, and its concentration ranged from 2.45 × 109 to 8.89 × 1010 copies in 1 gD. The abundance of the sul2 gene was also high, but its concentration ranged from 6.25 × 104 to 7.20 × 105 in 1 gD and was even five orders of magnitude lower in comparison with sul1.The gene sul1 in cattle manure digestate [sul2 in manure-fertilized soil [sul1 gene [sul1 and sul2 genes confer resistance by encoding forms of dihydropteroate synthase that are not sensitive to the drug [Other authors also reported high concentrations of igestate  and sul2zed soil . Sulfonaul1 gene . Howeverthe drug . These gthe drug , which sqepA gene ranged from 104 to 105 copies/gD in digestate samples. The concentration of the aac-(6′)-Ib-cr resistance gene was one order of magnitude lower, at 103 copies/gD in most samples. The concentration of the aac-(6′)-Ib-cr gene was one order of magnitude higher (104 copies/gD) only in sample BP1W, relative to the remaining samples. The aac-(6′)-Ib-cr gene encodes an enzyme which inactivates ciprofloxacin and norfloxacin by N-acetylation [qepA gene encodes efflux pump proteins of the major facilitator superfamily, which transport drugs to extracellular space. In the present study, the high abundance of qepA in digestate samples can be attributed to its non-specificity. The efflux pump encoded by qepA can decrease the toxic accumulation of antimicrobials inside cells, thus contributing to microbial growth during AD and promoting the transfer of ARGs between bacterial cells [The abundance of the tylation . The qepal cells .aac-(6′)-Ib-cr gene in digestate [qepA gene in sewage sludge [aac(6′)-Ib-cr and qepA genes in soil fertilized with digestate.Other authors also reported high abundance of the igestate , wastewaigestate , and rivigestate , as welle sludge . The preermF gene was abundant in all digestate samples, regardless of their origin, and its concentration ranged from 107 to 108 copies/gD. The ermF gene was most abundant (108 copies/gD) in samples of cattle manure digestate. These results suggest that digestate is a hot spot of ARGs. In the literature, strains of Enterococcus resistant to erythromycin were identified in ready-to-eat meat products, which implies that these genes can be transmitted to the gut microbiota of livestock and animal-based foods [The ed foods .cfxA was the most abundant beta-lactam-resistance gene, and its concentration reached 3.33 × 105 copies/g of digestate in sample BP1W (sewage sludge digestate).The gene 3 to 104 copies/gD) than in the remaining samples (105 copies/gD). Genes encoding resistance to beta-lactams are ubiquitous in the environment. The analyzed digestate samples were free of beta-lactams that could exert selection pressure, but they were characterized by high concentrations of beta-lactam-resistance genes that can be transported the soil environment with digestate, accumulate in plants and, consequently, reach the animals’ gut microbiota [All beta-lactam-resistance genes were also identified in digestate samples from BP3, which processes maize silage, slaughterhouse waste, potato pulp, and confectionary press cake (samples BP3S and BP3W), but their concentrations were one order of magnitude lower on average  were abundant in all digestate samples (regardless of their substrate composition), and their concentrations ranged from 4.37 × 105 to 5.84 × 107 copies/gD. These results could indicate that MGEs are involved in the spread of AR.Integrons participate in the spread of ARGs via HGT mediated by MGEs, and the r of HGT . In the intI1, intI2) were the first integrons to be strongly associated with MGEs. In other studies, sewage sludge and manure digestates frequently harbored intI1 [intI2 genes [Class 1 and 2 integrons  in digestate samples, depending on their origin. Heavy metals were identified in all samples, but not every digestate sample contained all of the analyzed HMs. Heavy metals such as Cd, Ni, Cr, Zn, and Hg were present in all samples. Lead was detected in sewage sludge digestate and digested mixtures of cattle manure and maize silage, but not in digestate originating from green waste. Sewage sludge digestate and digested mixtures of cattle manure and maize silage were characterized by similar HM levels (dm in sample BP2W (digested cattle manure and maize silage) to 830 mg/kg dm in sample BP1S.The presence and concentrations of the analyzed HMs differed significantly  and promote the spread of AR. According to previous research [Principal component analysis was used using all data from all samplings as variables to assess the overall patterns and variances of antibiotic concentrations, ARG, and HM levels in the BP digestate samples. The PCA produced three main data clusters . On the research , the preThe results of the PCA suggest that soil fertilization with digestate from agricultural BPs can increase the abundance of various ARGs, metal resistance genes (MRGs), drugs, drug residues and the associated microorganisms in the soil environment, and that digestate can significantly contribute to the spread of ARGs in the environment.Digestate is a rich source of organic matter and nutrients, and it is often used as organic fertilizer. However, digestate is also a vector of veterinary antibiotics, which are transmitted to the soil ecosystem and plants  because Abiotic factors can also contribute to the spread of ARGs. Physical factors such as wind, surface runoffs, flowing river water, and human activity can also increase AR in pathogenic bacteria in the environment ,86,87,88Despite the fact that digestate is the end product of the AD process, the analyzed digestate samples were characterized by high concentrations of ARGs and antibiotics. These observations suggest that digestate-based fertilizers should be applied at intervals longer than two months to restore background levels of ARGs in digestate-modified soils. This strategy would improve agricultural practices, inhibit the spread of ARGs, and decrease their concentrations in the environment ,93,94,95Effective strategies for controlling the use of antibiotics in agriculture are the simplest solution to reducing the concentrations of drugs and ARGs in cattle slurry, AD substrates, digestates, and farmland. Such strategies should be urgently implemented to minimize the health risks and adverse economic impacts of agricultural products containing drugs, ARB, and ARGs. Bioremediation and microbial electrolysis techniques are promoted in the agricultural sector to remove antibiotic residues and reduce AR. Heavy metals can be extracted from soil or wastewater with kaolin and sulfate-reducing bacteria. An integrated global strategy is needed to control the use of antibiotics in hospitals and the livestock sector, and novel methods should be introduced to optimize wastewater treatment, composting, and bioremediation of soil pollutants.Studies evaluating the presence and concentrations of various antibiotic classes and HMs in complex matrices, such as digested cattle manure, maize silage, sewage sludge, slaughterhouse waste, green waste, and confectionary press cake, pose an analytical challenge because different protocols have to be applied depending on digestate fraction and composition. Moreover, the composition of digestate samples can vary considerably because many agricultural BPs process mixed substrates with different proportions of plant biomass and manure. Industrial residues such as slaughterhouse waste also pose a considerable challenge for researchers. Despite these problems, this study demonstrated that most of the analyzed antibiotics and HMs were not degraded during the AD process. Digestate samples were highly contaminated with various drugs and HMs whose concentrations were influenced by the composition of the substrates processed by the examined BPs. Attention should also be paid to factors that contribute to the spread of AR in plant substrates. Unlike sewage sludge and cattle manure digestates, plant-based digestates did not contain drugs, but were characterized by high concentrations of many ARGs and selected HMs. Digestate can be regarded as a reservoir of ARGs due to the high abundance of various ARGs. Digestate samples were free of beta-lactams, but contained numerous genes encoding resistance to this class of antibiotics, which could point to high levels of AR already in the substrates. High concentrations of integrase genes also suggest that MGEs participate in the transmission of AR in digestate. The results of this study indicate that the use of digestate as organic fertilizer increases the risk of soil contamination with antibiotics, HMs, and ARGs.