Bv W Aloddi

SWEDEN Weibull ' Stockholm

A Statistical Distribution Function of Wide Applicability

This paper cl!iscusses the applicability of statistics to a wide field of problems. Examples of simple and complex distributions are given.

I

F a variable Xis attributed to the individuals of a population, the distribution function (df) of X, de no ted F(::r), may be defined as the number of all individuals having an X � x, divided by the total number of individuals. This function also give s t he pr ob abilit y P of choosing at random an individual having a value of X equal to or Jess than x, and thus \'!i'e have P(X 5 x) -F(z) .. .... .... ...... .. (I)

Any distribution function may be written in the form F(x) -I -e-"(z) ... .. .. ... ....... (2) This see ms to be a complication, but the advantage of thi s formal transformation depends on the relationship (l -P)• -e-,.. (z)_ ... .... .. .... . . . (3) The mer i ts of this formula will be demonstra�d on a simple problem.

Assume that we have a chain consisting of seve ral links. If we have foun d, by testing, the probability of failure Pat any load x applied to a "single" link, and if we want to find the probability of failure P. of a chain co nsisting of n links, we have to base our deductions upon the propos i tion th11t the ch11in as a whole hati failed, if any one of its parts has faile<l. Accordingly, the proba. bility of nonfailure of the chain, (I -P. ), is equal to the probability of the simultaneous nonfailure of all the links. Thus we have ( 1 -P ,. ) -(I -P)•. I f then the df of a single link takes the form Equation (2], we obtain P0 = I -e-n<p(z) . .. . . . . . . ... . . .. [4! Equ ation [4} gives the appropriate mathematical expression for the principle of the we akest link in the chain, or, more gen erally, for the si.ze effe ct on failures in solids.

The same method of reasoning may be applied to the large group of problems, I\ here the occurrence of an event in any part of an object may be said to have oocurred in the object. as a w ho le, e.g., the phenomena of yield limits, statical or dynamical strengths, electrical insula.tioo breakdowns, life of electric bulbs, or even death of man, aa the probRbility of surviving depends on the probability of not having died from many different c auses.

Xow we have to specify t he function 'P(x). The only neces-1 Professor at the Hoyal I nstitute or Technology; Scientific Adviser to AB Bofors; mai ling address Bofors, Sweden.

sary general condition this fun ction baa to satisfy is t0 be a posi tive, nonde creasin g function, vanishing at a value x., whi ch is not of necessity equal to zero.

The most simple function satisfying thill condition is

(x -x.)'"
To and thus we put F(x) "' le ............... {51

The only merit of this df is to be found in the fact that it i11 thP simplest mathematical express ion of the app ropriate form, Equa

ti on [2 ), which satisfies the necessary general conditions. Experi ence bas sh own that, in many cases, it fits the observations better than other known dist. ribution functions.

The obj ection has been stated that this distri but ion function has no theoretical basis. But in so far as the author understands, there are-with very few exceptions-the same objections against all other df, applied to real p o pulations from n atural o r b iologi cal fields, a.t lea.st in so far as the theoretical basis has any thing to do w ith the population in question. Furthermore, it is utterly hopeless to expect a theoretical basis for di stribu tion functions of mnd om variables such as strength properties of ma ter ials or of machine parts or particle sizes; the "particles" being fly ash, Cyrtoideae, or even adult males, born in the B ritish I sl e s.

It is be lieved that in such cases the only practicable way of progressing is to c ho ose a. simple function, test it empirically, and stick to it as long as none better hllll been found. lu accordance with this program the df Equ ation (5), has been applied not only to populations, for which it was originally intended, but also to populations from widely different fields, and, in many cases, with quite satisfactory results. The author has never been of the opinion that this function is always valid. On the contrary, he very much doubts the sense of speaking of the "correct" distri bution function, just as there is no meaning in asking for the correct strength values of an SAE steel, depending as it does, not only on the material itself, but nlso upon the manufacturer aud many other factors. In most cases, it is hoped that these factors will influence only the parameters. However, accidentally they may even affect the function itaclf. The purpose of this p aper has been to illustrate l\ith a few examples the experience that the df, Eq uatio n (5 ), may some times render good service.

The number of exampll's hns, hy space, been lim ited to t he follow i ng: The correctness of fit has been by applying the chi square method Of those populations, Nos. 1-3 are distributed in good agree ment with the df Equation [5 ], whereas the four remaining popu lations have to be split up into two components, before such an agreement is obtained. The first type will be caned a "simple" and the second type a "complex ... distribution.

The fundamental question now arises, whether this splitting up is a purely formnl operation, or whether it might unveil some hidden real causes. It may be said that any distribution may be represented by a sum of a sufficiently great number of simple distributions, just as an)' periodical function may be developed in a Fourier series. However, if the number of the components be small and the number of observations sufficiently 1 arge, the lik e- • lihood of rea.J causes seems to increase. In any case, it is very easy to produce real complex distributions by syntheses.

It seems obv i ous that the components of exampfos 4 and 5 are due to real causes. In examples 6 and 7 it is impossible to decide whether the division is a formal one or a real one, but the fact itself may be a valuable stimulus to a closer examination of th e observed material.

The specific data for the examples follow.

YIELD STRENGTH OF A BoFORS STEEL

The observed values are obtained a.s routine tests of a Bofors steel, the quality of which was chosen at random for purposes of demonstration only. Fig. 1 gives the curve and Table 1 the As a comparison, the values expected on the hypothesis of a normal distribution have been computed and are given in the last column of Table 1. If the classes 9-10 are pooled, the do ff are 8 -2 = 6. Then a x' = 18.17 gives a P = 0.008, which is not satisfactory at all.

. 4 • 1 -j! "' x..,• 38.57 kg/mm2 .0�1 �---1---1-� ---l ' ! x 0 • 7.74-kg/mm

SrzE DISTRIBUTION OF FLY ASH

The observed values are taken from J. M. Dalla Valle's work.' Fig. 2 gives the curve and Table 2 the

Size Distribution

of Fly-ash The observed values are taken from R. S. Koshal acd A. J.

N• 21 1 .2-1-l';" -+--+-r-+--�-�---1 1- 0 .8-2-="'o--+-_ _,.4'---! •u• 30µ
Turner.3 Fig. 3 gives the curve, and Table 3 the ; . 7-3 p. 0. 47

log (•-•u) 1-6 1-8 2.0 2-2 FIG. 4 LENGTH OF CYRTOIOEAE
In spite of the greater number of parameters, the fit of this dis tribu tion function is not as close as that of the first one.

LENGTH OF CYRTOIDEAE

This is the f i rst example of a complex distribution. The ob served values have been obtained from investigations by Dr. The number of individuals is too small for the x•-test.

FATIGUE LIFE OF A:<! ST-37 STEEL

The observed values are t.aken from Muller-Stock. 5 The fre quen.cy curve in Fig. 5• gives no impression of a complex dis tribution, which, on the other hand, may easily be seen when 164, 1949, p. 1047.

•"Der Einflu"s dau�rnd und unterbrochen wirkender, schwingender uberbcanspruchung auf die Bntwicklung des Daucrbruchs," by H.

Muli-er-Stoek, Mitteilunoen Kohle-und Eisen/orschuno, (March, 1938), by measurements from his Fi g. 13; reproduced in Fi g. 5 of this paper. 5 shows the close agreement between the observed and! the calculated values.

•0-0

• •O•I -I ! "'
•S-2 ! --i----t-,.'-.''"'---,.f-.'>--�

.o.t

•2 • 1 • B-1 Component I
x" • 4 .. The reason why this partition is so easily seen in Fig. 7 and not at all in Fig. 5, depends, of course, upon the much larger scatter in fatigue life than in yield strength. If claaaea 17-18 are pooled, the value of x' -4.50, and the dof f 9 -31/1 -51/t give a P • 0 . .56.

It ma.y be of interest to compare this reault with thoee of Chartier and Cram�.

Charlier says that, at the first look, the agree ment with the 11.ormal distribution aee ma very satiafaetory, but that a closer examination shows a small negative ekewneas and a small poei.

tive kurtosis.

Cram6r has calculated the valuea of x• on the hypotheeee of normal distribution and a.aymptotic expans i ons from it. The result waa a.a follows:

Normal distribution x' • 196.5 doff 13 P < 0.001 Firet approximation x' • 34.3 d of f 12 P < 0.001 Second appro:Dmation x' -14.9 doff n P • 0.19

The agreement is satisfactory in the third eaae only, requiring four terms Df the eeriee. Thia operation ia certainly of a purely formal character.

l

  Yie ld strength of a Bofors stet>) 2 Si�e distribution of fly Mh 3 Fiber st1-ength of Indinn cott.on 4 Leng t h of Cyrtoidcae 5 Fatigue life of a St-37 st.er.I In the Appendix: 6 Stature>s for adult mnles, born in th<-Briti�h !sit>� 7 Br<'adth of beans of Pha�e>olu� Vulgaris

  Fw. l YIELD STRENGTH oF A Bo.-oas STEEL values, observed and calculated. The parameters are x . = 38.57 kg/mm', x0 = 7.74 kg/mm', m = 2.934. Without pooling, the degrees of freedom (doff) arc 9-3 = 6. Then X' = 5.40 gives P = 0.49. The agreement is thus very satisfactory.

  values. The parameters are Xu = 30µ, xo = 128 µ, m = 2.288. Without pooling, the d off are 12-3 = 9. Then x' = 8.44 gives a P = 0.49. If the dasses 2-3 and 13-14 are pooled, the d of f are 7 and X' = 8.44 gives a p = 0.29.

  values. The parameters are Xu = 0.59 gram, xo = 3.73 grams, m = 1.456. If the classes 14 to 16 are pooled, the dof f are 13-3 =IO. Then x• = 11.45 gives a p = 0.35. The authors3 have pointed out that the most striking feature about the frequency curve is its asymmetry, showing a well marked predominance of weak fibers. It wa.s found-they say that the observation curve would be well fitted by a theoretical curve of Pearson's Type 1, having the following equation ( In this equation y represents the frequency of any strength x, e:x "Jlressed in grams. The values computed from this not very handy equation are shown in the last column of Table 3. The d off are 13 -5 = 8 (as there are 5 parameters). Then X2 -14.43 gives a P -0.07. '"Micromeritics," by J. M. Dalla Valle, Pitman Publishing Corporation. New York, N. Y., 1948, p. 57, Fig. 2. • '"Studies in the Sampling of Cotton fer the Determination of Fiber Properties," by R. S. Koshal :ind A. J. Turner. Journal of the Textile Institute Transactions, vol. 21, 1•930, pp. 325-370.

  Fig. 4 gives the curves and Table 4 the values of one of the complex populations. The undivided sample gives the curve marked N1 + Nz. It is easy to see that the distribution is a complex one, and that it is necessary to split up the population in two parts. By trial it was found that 86• of the individuals be longed to component No. I, and 14 to component No. 2. The parameters are: Component No. 1: x. = 3.75 µ, xo -63.2 µ, m = 2.097. Pooli11g the classes 2-3, 9-10, and 11-13 gives x' = 3.59. The do ff are 7 -3 = 4, and P = 0.47. Component No. 2: :i. = 122.0 µ, xo = 124.1 µ, m = 1.479.

•

  "A Statistical Analysi" of the Size of Cyrtoideae in Albatross Cores From the East Pacific Ocean," •by W. Weibull, Nature, vol.

  FIG. 5 FREQUENCY CURVE OF FATIGUE LIFE OF &r-37 ST&EL (Number of specimens versus number of stress cycles.)

FIG. 6

 6 FIG. 6 FATIGUE LIFE OF SN7 STEELIt may be pointed out that the frequency curve in Fig.5seems to be the result of a smoothin� operation on the cumulative frequency curve. Accordingly, the sampling errors of the ob served values in Table5 havebeen eliminated almost entirely (without affecting the function), which explains the really too good representation of the observed values. The real ca.uses • of this splitting up in two components may be found by examining the frequency curve of the yield strength of the same material, Fig. 7. It is easy to see that the material, probably not being killed,, is composed of two dif f erent kinds. If we suppose that all the specimens with a yield strength of less than 25 kg/mm• belong to Component No. 1, we obtain 14 speci mens out of 20, making 70 per cent. Exactly the same propor tion has been found by the statistical analysis, as 165/235 = 70 per cent.

FIG. 7

 7 FIG. 7 FREQUENCY CURVE OF YIELD STRENGTH OF ST•37 STEEL (Number or specimens versus yield .strength in 'kg/mm•.) If the classes 20-21 are pooled, x• = 5.11. The d of fare 8 3' ;, = 41 /2, which gives a P = 0.35. BREADTH OF BEANS OF PHASEOLUS VULGARIS This is a classical example, quoted from Charlier7 to exemplify the expansion in Edgeworth's series. If the population is divided into two parts, N, = 7900 and N,

TABLE l YIELD

 l STRENGTH OF A BOFORS STEEL

	1 2 3 4 5 6 7 8 9 10	" 32 33 34 35 36 37 38 39 40 42	Expected values n 10 36 84 150 224 291 340 369 383 389	Observed values n 10 33 81 161 224 289 336 369 383 389
		Yield Strength of a Bofors Stee l N • 389

Co:yield strength in 1.275 kg/mm•)

  (>:t"n.oile strength in graim)

			Expected Yalues n 118 64G 1232 1751 2161 2461 2667 2802 2886 2937 2966 2982 2991 2996 2999 3000	Observed ''ah.1es n 177 667 1219 1729 2153 2465 2664 2813 2887 2933 2962 2985 2991 299� 2999 3000		Pearson Type 1 " 127 659 1255 1777 2184 2480 2683 2816 2899 2949 2978 2994 3003 3007 3009 3010
			Fiber Strength of T ndian Cotton N• 3000
				w0 • 3 . 73gr:
		.9-1		.7	.9	I.I
	1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20	z 10 20 30 40 50 60 70 80 90 JOO 110 120 130 1, 10 150 160 170 180 190 200	m l 5 13 23 35 47 58 67 74 79 82 85 86 86 86 86 86 86 86 86	n1 4 7 9 II 12 13 14	Rl+l 1 5 13 23 35 47 58 67 74 79 82 85 86 90 93 95 97 98 99 100	values n1+s 0 5 12 24 38 45 58 69 70 80 82 84 86 90 93 95 97 98 99 JOO
				Xu• 3 . 75 ,o<
					2.097
				x2• 3.59

m • 1.4561 X.2 .all,4S;

i

•IO • 0.35 FIG. 3 FIBER STR>:NGTH 0>' INDIAN COTTON TABLE 4 LENGTH OF CYRTOIDEAE

(x = length in micron.'}) Observed �Expected values•--..

TABLE 5

 5 FATIGUE LlF'E OF ST-37 (Rotating-beam test at .,32 'kg/mm•)

	I 2 3 4 5 6 7 8 9 IO 11	N 10• 17.5 22.5 27.5 32.5 37.5 42. 5 47 . 5 52.5 57.5 62.5 67.5	�--Expeeted values---n1 nt nt.,.i 4.6 4.6 47 A 47 .4 125. I 125. l 161.2 8.1 169.3 164.9 28.0 192.9 165.0 41. g 206.9 165.0 51.0 216.0 165.0 57.0 222.2 165.0 61.0 226.0 165.0 63. 7 228.7 165 .0 65.6	Observed values n1.,.1 4.6 47 _4 125_ 1 169.2 192. 7 207 .3 215.9 222_2 225_9 228. 7

TABLE 7 PHASEOLUS

 7

	TABLE6	STATURES FOR ADULT MALES BORN IN THE BRITISH: ISLES
	"' 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77	"' 2 6 20 56 143 333 702 1350 2351 3641 4917 5787 6134 6197 6200 6200 6200 6200 6200 6200 6200	Expected values---n1 1&1.+1 2 6 20 56 143 333 702 1350 2351 36.ff 339 41117 6126 1079 7213 1671 7868 2039 8239 2233 8433 2324 8524 2363 8563 2378 8578 2383 8583 2385 8585	Observed v!!lues n1+1 2 6 20 61 144 313 707 1376 2366 3589 4918 6148 7211 7857 8249 8451 8530 8562 8578 8583 8585
	., 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18	'" 32 130 400 IOU 2145 3832 57l8 7140 7761 7890 7900 7900 7900 7900 7900 7900 7900 7900	nt "486 1525 2510 3229 3671 3908 4022 4071 409J 4098 4100	n1+1 32 130 400 1011 2145 3832 5718 7626 9286 10400 11129 11571 ll808 11922 11971 11991 11998 12000	n1+1 32 135 374 998 2185 3835 5718 7648 9286 10416 11153 11580 11801 11911 ll968 ll992 11998 12000
	6
	5
	4
	2
	I 22 0

{zheight in inches) VULGARIS (Breadth of bean• � 0.25 x + 6. 70 mm) O boerved �-Expected values valuea

'"An Introduction t!) the Theory of Statistics," by G. U. Yule an d M. G. Kendall, eleventh edition. J. B. Lippincott Com pany, Philadelphia. Pa., 1937, pp. 94 and 111.

Appendix

The foregoing statistical methods J1ave been applied to many problems outside the field of applied mechanics. It may perhaps be of interest to have examples of this kind, and for this reason, the following two are given with the tables only: If the classes 1-2 and 14-15 are pooled, we get x• = 11.80.

As we have 7 parameters altogether, (one of them is the partition of the population), we take 3'/, to each of the components. The do ff are then 12-3'/2 = 81/., which gives a P =• 0.20.

Component No. 2: x. = 67.4 in., xo = 2.3 in., m = 1.4662.


Arvind Varsani
email: arvind.varsani@asu.edu

Mart Krupovic

Family Genomoviridae: 2021 taxonomy update

The family Genomoviridae (phylum Cressdnaviricota, class Repensiviricetes, order Geplafuvirales) includes viruses with circular single-stranded DNA genomes encoding two proteins, the capsid protein and the rolling-circle replication initiation protein. The genomes of the vast majority of members in this family have been sequenced directly from diverse environmental or animal-and plant-associated samples, but two genomoviruses have been identified infecting fungi. Since the last taxonomic update of the Genomoviridae, a number of new members of this family have been sequenced. Here, we report on the most recent taxonomic update, including the creation of one new genus, Gemytripvirus, and classification of ~420 new genomoviruses into 164 new species. We also announce the adoption of the "Genus + freeform epithet" binomial system for the naming of all 236 officially recognized species in the family Genomoviridae.

The family Genomoviridae includes viruses with small circular single-stranded (ss) DNA genomes (~1.8-2.4 kb) encoding a rolling-circle replication initiation protein (Rep) and a capsid protein (CP) in an ambisense orientation [START_REF] Krupovic | Genomoviridae: a new family of widespread single-stranded DNA viruses[END_REF]. Whereas the genomoviral CP is not recognizably similar at the sequence level to the CPs of other known viruses, the Rep is homologous to those of other eukaryotic ssDNA viruses and is most similar to those of plant viruses of the family Geminiviridae, sharing several unique sequence motifs and forming a sister group in phylogenetic analyses [21][22][23]. Accordingly, the families Genomoviridae and Geminiviridae were included in the order Geplafuvirales [START_REF] Krupovic | Cressdnaviricota: a virus phylum unifying seven families of Rep-encoding viruses with singlestranded, circular DNA genomes[END_REF]. All eukaryotic ssDNA viruses encoding these related Reps, informally referred to as the CRESS DNA viruses [START_REF] Rosario | A field guide to eukaryotic circular single-stranded DNA viruses: insights gained from metagenomics[END_REF][START_REF] Zhao | Eukaryotic circular Rep-encoding single-stranded DNA (CRESS DNA) viruses: ubiquitous viruses with small genomes and a diverse host range[END_REF], were recently officially unified in the phylum Cressdnaviricota [START_REF] Krupovic | Cressdnaviricota: a virus phylum unifying seven families of Rep-encoding viruses with singlestranded, circular DNA genomes[END_REF][START_REF] Walker | Changes to virus taxonomy and the Statutes ratified by the International Committee on Taxonomy of Viruses (2020)[END_REF].

The founding member of the Genomoviridae [START_REF] Krupovic | Genomoviridae: a new family of widespread single-stranded DNA viruses[END_REF], Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), infects the phytopathogenic fungus Sclerotinia sclerotiorum [START_REF] Yu | A geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic fungus[END_REF] but can also replicate in its transmission vector, the mycophagous insect Lycoriella ingenua [START_REF] Liu | Fungal DNA virus infects a mycophagous insect and utilizes it as a transmission vector[END_REF]. However, the vast majority of genomoviruses have been discovered by metagenomics in diverse samples (see Supplementary Table S1), and the real extent of their host range remains unknown. In 2017, a sequence-based taxonomic framework was established for genomovirus classification [START_REF] Varsani | Sequence-based taxonomic framework for the classification of uncultured single-stranded DNA viruses of the family Genomoviridae[END_REF]. In particular, 78% genome-wide pairwise identity was chosen as a species demarcation threshold, whereas Rep sequence phylogeny was used to define genera. At the time, the family consisted of 121 members, which were classified based on genome sequences into 73 species divided into nine genera: Gemycircularvirus (43 species and 73 members), Gemyduguivirus (1 species and 1 member), Gemygorvirus (5 species and 9 members), Gemykibivirus (16 species and 29 members), Gemykolovirus (2 species and 3 members), Gemykrogvirus (3 species and 3 members), Gemykroznavirus (1 species and 1 member), Gemytondvirus (1 species and 1 member), and Gemyvongvirus (1 species and 1 member).

In the period since the establishment of the family Genomoviridae [START_REF] Krupovic | Genomoviridae: a new family of widespread single-stranded DNA viruses[END_REF] and the first report on the classification of then known genomoviruses (n = 122) [START_REF] Varsani | Sequence-based taxonomic framework for the classification of uncultured single-stranded DNA viruses of the family Genomoviridae[END_REF], ~420 new complete genome sequences of genomoviruses had Handling Editor: Sead Sabanadzovic. 1 3 been deposited in the GenBank database as of May 2020, including both virus isolates and viruses discovered by metagenomics [2, 3, 6-9, 11-16, 18, 24, 26-34, 37-42, 44-46, 48, 50-56, 58, 59, 61, 62, 64-69, 71, 73-81, 83]. Furthermore, the International Committee on Taxonomy of Viruses (ICTV) has recently adopted a freeform binomial species nomenclature, whereby virus species names have to consist of two words, with the first one being the genus name and the second being a free-form species epithet, which can consist of Latin letters and/or Arabic numerals [START_REF] Siddell | Binomial nomenclature for virus species: a consultation[END_REF]. All existing species that currently do not conform to this binomial format have to be renamed before 2023. Here, we report on the classification of the new genomoviruses as well as on other taxonomic changes in the family Genomoviridae, which were approved by the ICTV following the annual ratification vote in March of 2021.

The first notable change implemented in the Genomoviridae taxonomy was adoption of the binomial species nomenclature for all 73 existing species. The correspondence between the old and new binomial species names is shown in Table 1. We note that the names of viruses that are included in the corresponding species are not affected. For instance, the virus name Sclerotinia sclerotiorum hypovirulence associated DNA virus 1 remains unchanged although the species name Sclerotinia gemycircularvirus 1 has been changed to Gemycircularvirus sclero1.

The second taxonomic development involves the classification of ~420 new genomoviruses. We used the criteria of Rep amino-acid-based phylogeny (Fig. 1) to assign genomoviruses to genera, as outlined by Varsani and Krupovic [START_REF] Varsani | Sequence-based taxonomic framework for the classification of uncultured single-stranded DNA viruses of the family Genomoviridae[END_REF]. By contrast, the recently isolated Fusarium graminearum gemytripvirus 1 (FgGMTV1), infecting Fusarium graminearum, a fungal plant pathogen with worldwide distribution that causes Fusarium head blight (FHB) disease in wheat and barley [START_REF] Li | A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified virions[END_REF], formed a separate branch in the Rep phylogeny (Fig. 1). Notably, unlike other members of the Genomoviridae, which are monopartite, FgGMTV1 contains three genomic segments, each encoding a single protein [START_REF] Li | A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified virions[END_REF]: DNA-A encodes a Rep protein; DNA-B encodes a genomovirus-like CP; and DNA-C encodes a protein of unknown function. DNA-A and DNA-B are mutually interdependent for their replication, whereas DNA-C relies on DNA-A and DNA-B for replication and appears to enhance virus pathogenesis and transmission via conidia as well as accumulation of viral DNA in infected fungi [START_REF] Li | A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified virions[END_REF]. Phylogenetic analysis suggests that the multipartite genome of FgGMTV1 has evolved from a monopartite genome of an ancestral genomovirus. Thus, based on the Rep phylogeny and its multipartite genome organization, FgGMTV1 has been classified as a member of a new species, Gemytripvirus fugra1, within a new genus, Gemytripvirus (gemini-like myco-infecting tripartite virus) [START_REF] Li | A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified virions[END_REF].

Using the previously established species demarcation criteria [START_REF] Varsani | Sequence-based taxonomic framework for the classification of uncultured single-stranded DNA viruses of the family Genomoviridae[END_REF], namely, genome-wide pairwise identity of 78%, 35 viruses can be assigned to eight known species, while the remaining 389 viruses are classified to 164 new species (Table 1). The new species were named using the free-form binomial system. The greatly expanded dataset of genomoviruses has reinforced the validity of the previously established species demarcation criteria. Indeed, pairwise comparison of the representative sequences from each of the species (except for Gemytripvirus fugra1, whose members have a tripartite genome) showed that they share less than 78% genome-wide pairwise identity (Figs. 2 and3). A summary of the genome sequences of genomoviruses belonging to specific genera and their source are summarized in Figure 4, and additional details are provided in Supplementary Table S1. Accordingly, this threshold will continue to be used for further taxonomic classification of new genomoviruses.

Finally, we note that following the taxonomic assessment described herein, a number of new genomoviruses (n = 201; GenBank, download 15 May 2021) have been discovered [START_REF] Cibulski | A plate of viruses: viral metagenomics of supermarket chicken, pork and beef from Brazil[END_REF][START_REF] Cibulski | Detection of multiple viruses in oropharyngeal samples from Brazilian free-tailed bats (Tadarida brasiliensis) using viral metagenomics[END_REF][START_REF] De Nazare | Metagenomics of neotropical single-stranded DNA viruses in tomato cultivars with and without the Ty-1 gene[END_REF]25,[START_REF] Smith | Circular DNA viruses identified in short-finned pilot whale and orca tissue samples[END_REF]. Of these, 42 can be assigned to currently established species, and 161, once classified, are likely to represent new species and genera within the family Genomoviridae. However, we would like to discourage naming newly discovered viruses using official taxon names. For instance, an ssDNA virus infecting the phytopathogenic fungus Botrytis cinerea has been isolated recently and named Botrytis cinerea genomovirus 1 (BcGV1) [17]. Although BcGV1 displays a genomic organization similar to that of genomoviruses and encodes a related Rep, the CP encoded by this virus is unrelated to that of genomoviruses. Thus, placement of BcGV1 within the family Genomoviridae is questionable.

1 3

Gemycircularvirus furse1 Fur seal associated gemycircularvirus 1

Faecal-associated gemycircularvirus 4

KF371638

Gemycircularvirus geras1 Gerygone associated gemycircularvirus 1

Faecal-associated gemycircularvirus 6

KF371636

Gemycircularvirus geras2

Gerygone associated gemycircularvirus 2

Faecal-associated gemycircularvirus 5

KF371637

Gemycircularvirus geras3

Gerygone associated gemycircularvirus 3

Faecal-associated gemycircularvirus 3

Gemycircularvirus ptero1 Pteropus associated gemycircularvirus 1

Pacific flying fox faeces associated gemycircularvirus-10

KT732804

Gemycircularvirus ptero10 Pteropus associated gemycircularvirus 10

Pacific flying fox faeces associated gemycircularvirus-3

KT732794

Gemycircularvirus ptero2 Pteropus associated gemycircularvirus 2

Pacific flying fox faeces associated gemycircularvirus-2

KT732792

Gemycircularvirus ptero3 Pteropus associated gemycircularvirus 3

Pacific flying fox faeces associated gemycircularvirus-5

KT732797

Gemycircularvirus ptero4 Pteropus associated gemycircularvirus 4

Pacific flying fox faeces associated gemycircularvirus-13 KT732814 1 3

Gemycircularvirus ptero5 Pteropus associated gemycircularvirus 5

Pacific flying fox faeces associated gemycircularvirus-8

KT732801

Gemycircularvirus ptero6 Pteropus associated gemycircularvirus 6

Pacific flying fox faeces associated gemycircularvirus-9

KT732803

Gemycircularvirus ptero7

Pteropus associated gemycircularvirus 7

Pacific flying fox faeces associated gemycircularvirus-11

KT732807

Gemycircularvirus ptero8

Pteropus associated gemycircularvirus 8

Pacific flying fox faeces associated gemycircularvirus-14

KT732806

Gemycircularvirus ptero9

Pteropus associated gemycircularvirus 9

Pacific flying fox faeces associated gemycircularvirus-4 1 3

Unclassified virus (MK032746) Gemytondvirus

Fig. 1 Fig. 2

 12 Fig. 1 Maximum-likelihood phylogenetic tree of Rep amino acid sequences of 545 genomoviruses together with a subset of geminiviruses and an unclassified virus (MK032746) that are distantly related. The tree is rooted with geminivirus sequences (green) and that of MK032746, which is most closely related to classified members of the family Genomoviridae. Clades corresponding to different genomovirus genera are colored blue. The Rep sequence alignment was con-

Fig. 3

 3 Fig. 3 Pairwise distribution plot of the 236 representative sequences of genomoviruses (except Fusarium graminearum gemytripvirus 1 [species Gemytripvirus fugra1], which has a multicomponent genome), showing that no sequences from different species share >78% identity.

Fig.

  Fig.[START_REF] Cibulski | A plate of viruses: viral metagenomics of supermarket chicken, pork and beef from Brazil[END_REF] Summary of the number of genomovirids assigned to each genus and source from which the genomes was obtained

  P, Varsani A (2020) Diverse genomoviruses representing twenty-nine species identified associated with plants. Arch Virol 165:2891-2901 15. Halary S, Duraisamy R, Fancello L, Monteil-Bouchard S, Jardot P, Biagini P, Gouriet F, Raoult D, Desnues C (2016) Novel single-stranded DNA circular viruses in pericardial fluid of patient with recurrent pericarditis. Emerg Infect Dis 22:1839-1841 16. Hanna ZR, Runckel C, Fuchs J, DeRisi JL, Mindell DP, Van Hemert C, Handel CM, Dumbacher JP (2015) Isolation of a complete circular virus genome sequence from an alaskan black-capped chickadee (Poecile atricapillus) gastrointestinal tract sample. Genome Announc 3:e01081-15 17. Hao F, Wu M, Li G (2021) Characterization of a novel genomovirus in the phytopathogenic fungus Botrytis cinerea. Virology 553:111-116 18. Jia J, Fu Y, Jiang D, Mu F, Cheng J, Lin Y, Li B, Marzano SL, Xie J (2021) Interannual dynamics, diversity and evolution of the virome in Sclerotinia sclerotiorum from a single crop field. Virus Evol 7:veab032 19. Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods 14:587-589 20. Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform 20:1160-1166 21. Kazlauskas D, Dayaram A, Kraberger S, Goldstien S, Varsani A, Krupovic M (2017) Evolutionary history of ssDNA bacilladnaviruses features horizontal acquisition of the capsid gene from ssRNA nodaviruses. Virology 504:114-121 22. Kazlauskas D, Varsani A, Krupovic M (2018) Pervasive chimerism in the replication-associated proteins of uncultured singlestranded DNA viruses. Viruses 10:187 23. Kazlauskas D, Varsani A, Koonin EV, Krupovic M (2019) Multiple origins of prokaryotic and eukaryotic single-stranded DNA viruses from bacterial and archaeal plasmids. Nat Commun 10:3425 24. Kemenesi G, Kurucz K, Zana B, Foldes F, Urban P, Vlaschenko A, Kravchenko K, Budinski I, Szodoray-Paradi F, Bucs S, Jere C, Csosz I, Szodoray-Paradi A, Estok P, Gorfol T, Boldogh S, Jakab F (2018) Diverse replication-associated protein encoding circular DNA viruses in guano samples of Central-Eastern European bats. Arch Virol 163:671-678 25. Khalifeh A, Blumstein DT, Fontenele RS, Schmidlin K, Richet C, Kraberger S, Varsani A (2021) Diverse cressdnaviruses and an anellovirus identified in the fecal samples of yellow-bellied marmots. Virology 554:89-96

Table 1

 1 Nomenclatural and taxonomic changes in the family Genomoviridae

	Genus	New species name	Former species name	Exemplar virus	Accession no.
	Gemycircularvirus Gemycircularvirus abati1	-	Genomoviridae sp.	MK032723
		Gemycircularvirus alces1	-	Alces alces faeces associated	MG641204
				genomovirus MP84
		Gemycircularvirus alces2	-	Alces alces faeces associated	MG641207
				genomovirus MP157
		Gemycircularvirus ansal1	-	Genomoviridae sp.	MN928911
		Gemycircularvirus aspar1	-	Plant associated genomovirus 21	MH939436
		Gemycircularvirus austro1	-	Blackfly genomovirus 2	MK433234
		Gemycircularvirus austro2	-	Blackfly genomovirus 7	MK433239
		Gemycircularvirus austro3	-	Blackfly genomovirus 4	MK433236
		Gemycircularvirus austro4	-	Blackfly genomovirus 9	MK433241
		Gemycircularvirus austro5	-	Blackfly genomovirus 5	MK433237
		Gemycircularvirus austro6	-	Blackfly genomovirus 10	MK433242
		Gemycircularvirus bemta1	-	Bemisia-associated genomovirus	KY230614
				AdO
		Gemycircularvirus blabi1	Blackbird associated gemycircular-	Faecal-associated gemycircularvirus	KF371643
			virus 1	1a
		Gemycircularvirus bovas1	Bovine associated gemycircularvi-	Faeces associated gemycircularvirus	KT862253
			rus 1	22
		Gemycircularvirus bromas1 Bromus associated gemycircular-	Bromus-associated circular DNA	KM510192
			virus 1	virus 3
		Gemycircularvirus canlup1	-	Lupine feces-associated gemycircu-	KY214442
				larvirus 2
		Gemycircularvirus cassa1	Cassava associated gemycircular-	Cassava associated cicular DNA	JQ412056
			virus 1	virus
		Gemycircularvirus chicas1	Chicken associated gemycircular-	Faeces associated gemycircularvirus	KT862243
			virus 1	20
		Gemycircularvirus chicas2	Chicken associated gemycircular-	Faeces associated gemycircularvirus	KT862242
			virus 2	17
		Gemycircularvirus chickad1 Chickadee associated gemycircular-	Poecile atricapillus GI tract-associ-	KT309029
			virus 1	ated gemycircularvirus
		Gemycircularvirus citas1	-	Citrus Tunisia genomovirus 2	MN708485
		Gemycircularvirus cybusi1	-	Spider associated circular virus 1	MH545503
		Gemycircularvirus denbre1	-	Bark beetle-associated genomovi-	MG571096
				rus 1
		Gemycircularvirus denbre2	-	Bark beetle-associated genomovi-	MG571098
				rus 2
		Gemycircularvirus denbre3	-	Bark beetle-associated genomovi-	MG571099
				rus 3
		Gemycircularvirus denbre4	-	Bark beetle-associated genomovi-	MG571100
				rus 4
		Gemycircularvirus denpo1	-	Bark beetle-associated genomovi-	MG571101
				rus 5
		Gemycircularvirus derva1	-	Tick-associated genomovirus 1	MF173065
		Gemycircularvirus dichism1 -	Plant associated genomovirus 19	MH939446
		Gemycircularvirus draga1	Dragonfly associated gemycircular-	Dragonfly-associated circular virus	JX185429
			virus 1	2
		Gemycircularvirus echiam1 -	Thrips-associated genomovirus 1	KY308268
	-	Gemycircularvirus equas1	Equine associated gemycircularvi-	Faeces associated gemycircularvirus	KT862248
			rus 1	18
		Gemycircularvirus erati1	-	Giant house spider associated circu-	MH545509
				lar virus 1
		Gemycircularvirus euhet1	-	Euphorbia heterophylla associated	MH047858
				gemycircularvirus

1 3 Table 1

 31

	(continued)
	Genus	New species name	Former species name	Exemplar virus	Accession no.

1 3 Table 1

 31

	(continued)
	Genus	New species name	Former species name	Exemplar virus	Accession no.
		Gemycircularvirus malas1	Mallard associated gemycircular-	Faecal-associated gemycircularvi-	KF371635
			virus 1	rus 7
		Gemycircularvirus minio1	Miniopterus associated gemycircu-	Bat circovirus	KJ641719
			larvirus 1
		Gemycircularvirus miniti1	-	Genomoviridae sp.	MK032736
		Gemycircularvirus miniti2	-	Genomoviridae sp.	MK032701
		Gemycircularvirus miniti3	-	Genomoviridae sp.	MK032712
		Gemycircularvirus miniti4	-	Genomoviridae sp.	MK032709
		Gemycircularvirus minti6	-	Genomoviridae sp.	MH617534
		Gemycircularvirus mocha1	-	Momordica charantia associated	MH047857
				gemycircularvirus
		Gemycircularvirus monas1	Mongoose associated gemycircular-	Mongoose feces-associated gemycir-	KP263547
			virus 1	cularvirus d
		Gemycircularvirus mosqi1	Mosquito associated gemycircular-	Mosquito VEM virus SDBVL G	HQ335086
			virus 1
		Gemycircularvirus mouti1	-	Genomoviridae sp.	MK032728
		Gemycircularvirus mouti10	-	Genomoviridae sp.	MK032705
		Gemycircularvirus mouti11	-	Genomoviridae sp.	MK032718
		Gemycircularvirus mouti12	-	Genomoviridae sp.	MK032702
		Gemycircularvirus mouti2	-	Genomoviridae sp.	MK032755
		Gemycircularvirus mouti3	-	Genomoviridae sp.	MK032715
		Gemycircularvirus mouti4	-	Genomoviridae sp.	MK032719
		Gemycircularvirus mouti5	-	Genomoviridae sp.	MK032753
		Gemycircularvirus mouti6	-	Genomoviridae sp.	MK032735
		Gemycircularvirus mouti7	-	Genomoviridae sp.	MK032738
		Gemycircularvirus mouti8	-	Genomoviridae sp.	MK032752
		Gemycircularvirus mouti9	-	Genomoviridae sp.	MK032714
		Gemycircularvirus odona1	Odonata associated gemycircular-	Odonata associated gemycircular-	KM598385
			virus 1	virus-1
		Gemycircularvirus odona2	Odonata associated gemycircular-	Odonata associated gemycircular-	KM598387
			virus 2	virus-2
		Gemycircularvirus oltre1	-	Olive associated gemycircularvirus	MH444690
				1
		Gemycircularvirus opunt1	-	Plant associated genomovirus 25	MK947372
		Gemycircularvirus oxcor1	-	Oxalis corniculata genomoviridae	MN823668
		Gemycircularvirus pleca1	-	Gemycircularvirus sp.	KY302866
		Gemycircularvirus poass1	Poaceae associated gemycircular-	Poaceae-associated gemycircular-	KT253577
			virus 1	virus 1
		Gemycircularvirus porci1	Porcine associated gemycircular-	Faeces associated gemycircularvirus	KT862250
			virus 1	19
		Gemycircularvirus porci2	Porcine associated gemycircular-	Faecal-associated gemycircularvi-	KF371640
			virus 2	rus 2

Table 1

 1

	(continued)
	Genus	New species name	Former species name	Exemplar virus	Accession no.

Table 1

 1

	KT732795

1 3 Table 1

 31

	(continued)
	Genus	New species name	Former species name	Exemplar virus	Accession no.
		Gemykibivirus hydro1	-	Capybara genomovirus 4	MK483076
		Gemykibivirus hydro2	-	Capybara genomovirus 2	MK483073
		Gemykibivirus hydro3	-	Capybara genomovirus 12	MK483084
		Gemykibivirus minti1	-	Genomoviridae sp.	MK032742
		Gemykibivirus monas1	Mongoose associated gemykibivirus	Mongoose feces-associated gemycir-	KP263545
			1	cularvirus b
		Gemykibivirus mouti1	-	Genomoviridae sp.	MK032703
		Gemykibivirus mouti2	-	Genomoviridae sp.	MK032748
		Gemykibivirus pitis1	-	Genomoviridae sp.	MK032749
		Gemykibivirus pitis2	-	Genomoviridae sp.	MK032721
		Gemykibivirus planta1	-	Plant associated genomovirus 2	MH939363
		Gemykibivirus planta2	-	Plant associated genomovirus 29	MK947376
		Gemykibivirus ptero1	Pteropus associated gemykibivirus 1 Pacific flying fox faeces associated	KT732813
				gemycircularvirus-12
		Gemykibivirus raski1	-	Genomoviridae sp.	MK032704
		Gemykibivirus rhina1	Rhinolophus associated gemykibi-	Bat circovirus	KJ641737
			virus 1
		Gemykibivirus rhina2	Rhinolophus associated gemykibi-	Bat circovirus	KJ641726
			virus 2
		Gemykibivirus sewopo1	Sewage derived gemykibivirus 1	Sewage-associated gemycircular-	KJ547643
				virus-3
		Gemykibivirus sewopo2	Sewage derived gemykibivirus 2	Sewage-associated gemycircular-	KJ547642
				virus-2
		Gemykibivirus turti1	-	Genomoviridae sp.	MK012443
		Gemykibivirus waste1	-	Genomoviridae sp.	MT309857
		Gemykibivirus womot1	-	Genomoviridae sp.	MK032756
	Gemykolovirus	Gemykolovirus abati1	-	Genomoviridae sp.	MK032717
		Gemykolovirus citas1	-	Citrus Tunisia genomovirus 1b	MN708482
		Gemykolovirus derva1	-	Tick-associated genomovirus 2	MF173066
		Gemykolovirus easlu1	-	Grasshopper associated circular	MH545499
				virus 1
		Gemykolovirus echia1	-	Thrips-associated genomovirus 4	KY308270
		Gemykolovirus gopha1	-	Tortoise genomovirus 9	MK570209
		Gemykolovirus gopha2	-	Tortoise genomovirus 13	MK570213
		Gemykolovirus hadtis1	-	Genomoviridae sp.	MK032747
		Gemykolovirus heris1	-	Plant associated genomovirus 7	MH939374
		Gemykolovirus lepam1	-	Alces alces faeces associated	MG641203
				genomovirus MP68
		Gemykolovirus poaspe1	-	Plant associated genomovirus 9	MH939382
		Gemykolovirus prupe1	-	Amygdalus persica genomoviridae	MN823669
		Gemykolovirus ptero1	Pteropus associated gemykolovirus 1 Pacific flying fox faeces associated	KT732798
				gemycircularvirus-6
		Gemykolovirus ptero2	Pteropus associated gemykolovirus 2 Pacific flying fox faeces associated	KT732800
				gemycircularvirus-7
		Gemykolovirus segpa1	-	Tubeweb spider associated circular	MH545501
				virus 1
		Gemykolovirus troti1	-	Genomoviridae sp.	MK032739
	Gemykrogvirus	Gemykrogvirus abati1	-	Genomoviridae sp.	MK032716
		Gemykrogvirus apime1	-	Apis mellifera genomovirus 2	MH973741
		Gemykrogvirus bovas1	Bovine associated gemykrogvirus 1 HCBI9.212 virus	LK931484
		Gemykrogvirus carib1	Caribou associated gemykrogvirus 1 Caribou feces-associated gemycir-	KJ938717
				cularvirus

1 3 Table 1

 31

	(continued)
	Genus	New species name	Former species name	Exemplar virus	Accession no.
		Gemykrogvirus galga1	-	Chicken genomovirus mg4_1247	MN379609
		Gemykrogvirus galga2	-	Chicken stool-associated gemycir-	KY056250
				cularvirus
		Gemykrogvirus galga3	-	Chicken genomovirus mg4_1173	MN379605
		Gemykrogvirus galga4	-	Chicken genomovirus mg7_78	MN379614
		Gemykrogvirus galga5	-	Chicken genomovirus mg4_1165	MN379604
		Gemykrogvirus giapa1	-	Giant panda associated gemycircu-	MF327559
				larvirus
		Gemykrogvirus hadtis1	-	Genomoviridae sp.	MK032724
		Gemykrogvirus humas1	-	Gemycircularvirus sp.	MT649486
		Gemykrogvirus sewopo1	Sewage derived gemykrogvirus 1	Sewage-associated gemycircular-	KJ547634
				virus-4
	Gemykronzavirus	Gemykronzavirus anima1	-	Genomoviridae sp.	MK032727
		Gemykronzavirus haeme1	-	Finch associated genomovirus 8	MK249245
		Gemykronzavirus hydro1	-	Capybara genomovirus 10	MK483082
		Gemykronzavirus poaspe1	-	Plant associated genomovirus 24	MH939435
		Gemykronzavirus rabas1	Rabbit associated gemykroznavirus	Faecal-associated gemycircularvirus	KF371631
			1	11
		Gemykronzavirus solas1	-	Plant associated genomovirus 23	MH939385
		Gemykronzavirus zizan1	-	Zizania latifolia genomoviridae	MN823671
	Gemytondvirus	Gemytondvirus ostri1	Ostrich associated gemytondvirus 1 Faecal-associated gemycircularvirus	KF371630
				12
	Gemytripvirus	Gemytripvirus fugra1	-	Fusarium graminearum gemytrip-	MK430076,
				virus 1	MK430077,
					MK430078
	Gemyvongvirus	Gemyvongvirus humas1	Human associated gemyvongvirus 1 Human plasma-associated gemycir-	KP974693
				cularvirus
	Gemyvongvirus	Gemyvongvirus minit1	-	Genomoviridae sp.	MK032710
		Gemyvongvirus minit2	-	Genomoviridae sp.	MK032740

4

  Summary of the number of genomovirids assigned to each genus and source from which the genomes was obtained circular DNA virus with similarities to Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1. Arch Virol 159:1527-1531 13. Fontenele RS, Lacorte C, Lamas NS, Schmidlin K, Varsani A, Ribeiro SG (2019) Single stranded DNA viruses associated with capybara faeces sampled in Brazil. Viruses 11:710 14. Fontenele RS, Roumagnac P, Richet C, Kraberger S, Stainton D, Aleamotu'a M, Filloux D, Bernardo P, Harkins GW, McCarthy J, Charles LS, Lamas NS, Abreu EFM, Abreu RA, Batista GB, Lacerda ALM, Salywon A, Wojciechowski MF, Majure LC, Martin DP, Ribeiro SG, Lefeuvre

		Actinopterygii	Arachnida	Aves	Embryophyte	Environmental	Gastropoda	Insecta	Leotiomycetes	Mammalia	Reptilia	Total
	Gemycircularvirus	8	10 49	35	1 3	2	28	3	94	9	251
	Gemyduguivirus	2		1	12			3		4		22
	Gemygorvirus			3	8	1				6		1 8
	Gemykibivirus	3	1	54	17	6	2	10		46	15	154
	Gemykolovirus	2	2		46		1	2		5	2	6 0
	Gemykrogvirus	1		1 2		1	1	1		5	1	2 2
	Gemykronzavirus			3	7					3		13
	Gemytondvirus			1								1
	Gemytripvirus								1			1
	Gemyvongvirus	2								1		3
	Total	18 13 123	125	2 1	6	44	4 164	27 545
	1 3

Acknowledgements M.K. was supported by l'Agence Nationale de la Recherche (grant ANR-20-CE20-0009-02) and Ville de Paris (Emergence(s) project MEMREMA).

Family Genomoviridae: 2021 taxonomy update 1 3

Supplementary Information

The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s00705-021-05183-y.

Declarations

Conflict of interest The authors declare no conflicts of interest.

Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Louis Stéphane

Le Clercq

Desiré Lee

Antoinette Kotzé

Molecular age estimation based on promotor CpG methylation using Methylation Sensitive PCR

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Chronological age is a key factor in animal ecology, as many biological traits emerge and change over time. Such traits include: development, age of reproductive maturity, reproductive success, future reproductive potential, and mortality. Several molecular methods have emerged as potential vehicle for biological age determination. The aim of this experiment is to ascertain if a Methylation Sensitive PCR (MSP) could be developed to screen for methylation at a previously identified site in the GRIA2 gene.

Methods

•

MSP was conducted with the EpiScope® MSP kit on the OneStep Plus real-time instrument following manufacturers' instructions, increasing the 1 st denaturation to 1 min. • Primers were designed by eye for both methylated and unmethylated target CpG's in the GRIA2 promoter identified in previous studies using PyroMark sequencing. • EpiTect® PCR Control DNA was used to assay the specificity and selectivity of the methylated and unmethylated primer sets.

Results

The primers designed for the GRIA2 CpG was able to amplify the selected CpG with great efficiency making MSP a promising method of methylation screening. However, primer design to assay a specific site faces many problems and selectivity for methylated vs. unmethylated may not be achievable.

• After initial optimization, the assay was able to successfully amplify the unmethylated control DNA with an efficiency of 97.6% and R²=99% (Fig. 1).

•

Strong amplification plots (Fig. 2) were observed for a range of 0.3-20 ng input DNA.

•

The melting curve (Fig 3 .) showed a characteristic mixture of two products, with a single peak for the product. • Identical results were obtained for methylated DNA. (1) Research and Scientific Services, National Zoological Gardens of South Africa (SANBI), Pretoria.

Discussion & Conclusion

(2) Department of Genetics, Faculty of Natural and Agricultural Sciences, University of the Free State, Bloemfontein.

Figure 1 :

 1 Figure 1: Standard curve of unmethylated control DNA.Figure 2: Amplification plot of control DNA (0.3-20 ng).

Figure 2 :

 2 Figure 1: Standard curve of unmethylated control DNA.Figure 2: Amplification plot of control DNA (0.3-20 ng).

Figure 3 :

 3 Figure 3: Melting curve of unmethylated DNA.