Experimental evolutionary studies on the genetic autonomy of the cytoplasmic genome “plasmon” in the Triticum (wheat)–Aegilops complex

Koichiro Tsunewakia,1,2, Naoki Morib, and Shigeo Takumic,1

aGenetics, Kyoto University, 606-8502 Kyoto, Japan; bCrop Evolution, Graduate School of Agricultural Science, Kobe University, 657-8501 Kobe, Japan; and cPlant Genetics, Graduate School of Agricultural Science, Kobe University, 657-8501 Kobe, Japan

Contributed by Koichiro Tsunewaki, December 27, 2018 (sent for review October 3, 2018; reviewed by Bikram S. Gill and Ronald L. Phillips) The term “plasmon” is used to indicate the whole cytoplasmic been useful for elucidating their genetic relatedness and di- genetic system, whereas “genome” refers to the whole nuclear versification (7). Because the plasmon is maternally transmitted genetic system. Although maternal inheritance of the plasmon is and its genetic characteristics remain unchanged for long pe- well documented in angiosperms, its genetic autonomy from the riods of evolutionary time, as revealed in the present in- coexisting nuclear genome still awaits critical examination. We vestigation, plasmon analysis yields critical information on the tested this autonomy in two related studies: One was to deter- maternal lineage of a species in contrast to genome analysis, mine the persistence of the genetic effect of the plasmon of Aegi- which gives information on the parent–offspring relationship lops caudata (genome CC) on the phenotype of common wheat, (8, 9); therefore, combination of the genome and plasmon Triticum aestivum strain “Tve” (genome AABBDD), during 63 y analyses is useful in elucidating the maternal and paternal (one generation per year) of repeated backcrosses of Ae. caudata lineages of a species. and its offspring with pollen of the same Tve wheat, and the To make a comparative analysis of the phenotypic effects of a second was to reconstruct an Ae. caudata strain from the genome plasmon, it is necessary to select suitable genotypes for their of this strain and its plasmon that had been resident in Tve wheat expression. We selected a set of genotypes that were predicted to for 50 generations, and to compare the phenotypic and organellar give viable offspring in the cross between them and the plasmon GENETICS DNA characteristics between the native and reconstructed strains. donor(s) in the hope of producing alloplasmic lines with the Results indicated no change in the effect of Ae. caudata plasmon former’s genome and the latter’s plasmon by the repeated on Tve wheat during its stay in wheat for more than half a cen- backcross method of Correns (10), which was applied first by tury, and no difference between the native and reconstructed Correns to the genus Cirsium and later by Michaelis (11) to caudata strains in their phenotype and simple sequence repeats Epilobium. In the Triticum–Aegilops complex, Kihara (12) stud- in their organellar DNAs, thus demonstrating the prolonged ge- ied two alloplasmic lines, Aegilops aucheri (synonymous to netic autonomy of the plasmon from the coexisting genomes of Aegilops speltoides var. aucheri) having the Aegilops longissima wheat and several other species that were used in the reconstruc- plasmon up to the fifth substitution backcross (SB) generation tion of Ae. caudata. The relationship between the proven genetic and Triticum aestivum with the Aegilops caudata plasmon up to autonomy of the plasmon under changing nuclear conditions and the 10th SB generation, both of which were produced by re- its diversification during evolution of the Triticum–Aegilops com- peated SBs of the respective F1 hybrids. plex is discussed. Here, we used this method for producing the materials neces- sary to test the plasmon’s genetic autonomy by two interconnected Triticum–Aegilops complex | plasmon autonomy | species reconstruction | alloplasmic wheat | maternal lineage Significance “ ” he term plasmon indicates the whole cytoplasmic genetic In angiosperms, nuclear genes are inherited from both parents, Tsystem, comprising chloroplast and mitochondrial genomes, “ ” following Mendelian laws, whereas genes in the two cyto- whereas the term genome is used to indicate the whole nuclear plasmic organelles, the chloroplast and mitochondrion, are genetic system (1, 2). Maternal inheritance of the plasmon is well usually transmitted only through the female parent. In this documented in angiosperms (3, 4), but its genetic autonomy, study, we show experimentally that the cytoplasmic organellar namely, the independence of the plasmon from the coexisting genes of Aegilops remained autonomous from coexisting nu- genome, still needs critical examination. Although the chloro- clear genes of wheat, in their replication and transmission, for plast and mitochondrion both possess their own genome, their more than half a century. We also infer that the cytoplasmic functions are often expressed by interaction between their genes genome “plasmon” has retained genetic autonomy during and those of the coexisting nucleus. For example, the chloroplast 0.5 My of evolution of the Triticum–Aegilops complex, indicating enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) its usefulness in tracing the maternal lineage of a species. consists of two kinds of subunits, small and large RuBisCOs, which are encoded by the nuclear multigene family rbcS and the chloroplast Author contributions: K.T. designed research; K.T. and N.M. performed research; K.T. and gene rbcL, respectively (5). Similarly, the mitochondrial enzyme S.T. analyzed data; K.T. and S.T. wrote the paper; and N.M. performed molecular analysis cytochrome c oxidase consists of 13 subunits, of which 10 and of cytoplasmic DNAs. three are encoded by nuclear and mitochondrial genes, re- Reviewers: B.S.G., Kansas State University; and R.L.P., University of Minnesota. spectively (6). Thus, the genetic autonomy of the plasmon does The authors declare no conflict of interest. not mean independence of its function from that of genome Published under the PNAS license. but, rather, independence of its replication and transmission 1To whom correspondence may be addressed. Email: [email protected] or takumi@ from control by the nuclear genome. kobe-u.ac.jp. The plasmon is characterized experimentally by two methods: 2Present address: Kasugadai 6-14-10, Nishi-ku, Kobe, 651-2276 Hyogo, Japan. DNA analysis of organellar genes or genomes and analysis of its This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. genetic effects on morphology, physiology, and other traits of 1073/pnas.1817037116/-/DCSupplemental. host plants. Comparative analysis of plasmons in related taxa has

www.pnas.org/cgi/doi/10.1073/pnas.1817037116 PNAS Latest Articles | 1of9 Downloaded by guest on September 29, 2021 studies. The first was to examine persistence of the genetic effects Production of an alloplasmic Tve strain with the Ae. caudata plasmon. of the plasmon of Ae. caudata var. polyathera (2n = 14, genome Throughout the present investigations, all materials of wheat and CC; synonymous to Triticum dichasians) on the phenotype of a Aegilops were grown at one generation per year. In 1949, Kihara common wheat variety, T. aestivum var. erythrospermum (2n = 42, (15) crossed Ae. caudata polyathera as the female to Tve wheat. “ ” AABBDD; strain Tve ), during 63 generations (i.e., 63 y) of He successively backcrossed this F1 hybrid and its later back- repeated backcrosses of the F hybrid, Ae. caudata var. polyathera 1 crossed progeny with pollen of Tve wheat until the SB16 gener- (\) × T. aestivum var. erythrospermum (_) and its offspring with ation. SBs of this alloplasmic wheat, (caudata)-Tve SB16, with pollen of Tve wheat, testing the persistence of the genetic effect pollen of the same wheat were continued by Tsunewaki (7) and of the Ae. caudata plasmon during 63 generations of coexistence the present study up to the SB generation in 2016. with the wheat genome. The second study was to reconstruct Ae. 63 Male sterility expression. Seed fertilities expressed as the seed set- caudata var. polyathera strain KU6-1 from the genome of a native ting rates obtained by self-pollination and manual backcrossing Ae. caudata var. polyathera strain and its plasmon, which had resided in common wheat for 50 generations and in seven other of the F1 and its backcrossed progeny with the pollen of Tve strains for 12 additional generations, proving no detectable ge- wheat, up to the SB63 generation of (caudata)-Tve, are shown in netic difference between the native and reconstructed Ae. caudata Fig. 1A, in which gray circles on a dotted line and open circles on strains, including their organellar DNAs. The results obtained from a solid line represent the seed setting rates by self-pollination these studies demonstrated the genetic autonomy of the Ae. caudata and backcrossing, respectively. plasmon from coexisting alien genomes for more than half a cen- Data for the backcrossing in two successive generations, SB15 tury. Finally, we wanted to know how long the observed genetic (1964) and SB16 (1965), made by Kihara were not available. In autonomy of the plasmon can be retained in evolution, for which addition, the backcross of (caudata)-Tve was not made in 1982, purpose we examined our previous data on plasmon differentia- 1983, 2002, or 2004, causing four 1-y gaps between the initial cross tion in the evolution of the Triticum–Aegilops complex; this point is and the following SB generations; thus, there were 64 generations considered in Discussion. in total in the 68 calendar years from 1949 to 2016. Because the backcrossed seed fertility fluctuated widely Results among the SB generations due to the small number of hand- Persistence of the Genetic Effects of Ae. caudata Plasmon During 63 pollinated florets per generation, linear regression of the back- Generations of Repeated Backcrosses with Pollen of the Common crossed seed fertility (%; Y) against the SB generation (X) was Wheat Strain Tve. We first explain the designation of the strains we used. Kimber and Tsunewaki (13) proposed species names calculated for 60 SB generations, from SB2 to SB63, excluding and their genome and plasmon types for all species of the Tri- two generations of unavailable data. The linear regression ticum–Aegilops complex, a system to which we adhere with two obtained is shown in Fig. 1A: minor modifications. First, whereas their taxonomic system = + ð Þ combined two genera, Triticum and Aegilops, into the single ge- Y 0.241X 60.7 % nus “Triticum,” we separate the two genera. Second, we use italics to indicate plasmon type, so as to distinguish the plasmon from the nuclear genome. We produced 552 alloplasmic wheat strains from all possible combinations between 46 alien plasmons and 12 genotypes of common wheat (14). We developed the following unified system for their designation: An alloplasmic line having the genome and plasmon derived from different species (or strains) is indicated by a formula consisting of the name of the plasmon donor in parentheses and the species name or genome symbol of the ge- nome donor, connecting the two names with a hyphen. When necessary, to indicate genetic homozygosity of the genome, the number of backcrosses made with the same recurrent pollen parent is given by a subscript after SB (where “substitution” means nucleus substitution) or by a superscript after the name of the genome donor. For example, an alloplasmic line of Tve wheat with the Ae. caudata plasmon at its 50th backcross gen- eration is designated as either (caudata)-Tve SB50 or (caudata)- Tve51, with “51” being the sum of the first cross producing the initial F1 hybrid and the following 50 repeated backcrosses, in all of which the same genome donor was used as the pollen parent. Next, an Ae. caudata plasmon that stayed for 50 generations or more in Tve wheat is designated “caudataTve plasmon” when necessary to distinguish it from the native caudata plasmon of the variety typica or polyathera, which are designated caudatat or caudatap, respectively. Hereafter, “var.” is omitted, and varieties are written simply as Ae. caudata typica or Ae. caudata polyathera. We distinguish between two or three homologous genomes existing in different species or varieties by adding the initial of Fig. 1. Seed and pollen fertilities and germless grain production of (cau- their taxonomic name as a superscript to their genome symbol: data)-Tve strain. (A) Seed fertilities from the F1 to SB63 generations obtained p t c Ae. caudata by self-pollination and repeated backcrossing with pollen of Tve wheat. The Examples are C ,C, and C for the C genome of starting point for reconstruction of the Ae. caudata polyathera strain is polyathera, Ae. caudata typica, and Aegilops cylindrica, re- c indicated by the red arrow. Pollen grains of normal Tve wheat (B)and spectively. Finally, we use the symbol D for the D genome of Ae. its alloplasmic (caudata)-Tve SB63 plant (C) are shown. (Scale bars: B and C, cylindrica, leaving the basic symbols A, B, and D of the three 100 μm.) (D) Normal (Left) and germless (Right) grains produced by artificial

genomes of common wheat without any superscript. pollination of (caudata)-Tve SB61–63 plants with normal Tve pollen.

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1817037116 Tsunewaki et al. Downloaded by guest on September 29, 2021 The average backcrossed seed fertility of the 60 SB generations rather than an effect of the plasmon, because no such change was 68.7%; this figure indicated female fertility of (caudata)-Tve occurred on three other internode lengths. Thus, we conclude as being almost normal. The regression coefficient of b = 0.241 ± that the genetic effect of the Ae. caudata polyathera plasmon on 0.048 was statistically significant at the 5% level of probability for the phenotype of Tve wheat is limited to male sterility induction df = 58, with 95% fiducial limits of 0.193–0.289. This means a and germless grain production, which did not change during 0.241% improvement in female fertility by each backcross, in- 63 repeated backcross generations. clusive of all possible effects, such as the different environments in which backcrosses were made, namely, Mishima (F1 to SB16), Reconstruction of the Ae. caudata polyathera Strain from the Kyoto (SB17 to SB40), Fukui (SB41 to SB51), and Kobe (SB52 Genome of Native polyathera Strain and Its Plasmon Resident for to SB63). 50 Generations in Common Wheat. The entire procedure for In contrast, the selfed seed setting of (caudata)-Tve was zero reconstructing Ae. caudata polyathera consisted of five stages, as in all backcross generations, except for three (SB3,SB12, and depicted in Fig. 2A. Modal chromosome configurations at the SB26) in which one to three seeds were set in a total of two to first meiotic metaphase (MI) of pollen mother cells (PMCs; Fig. 10 ears covered by paper bags before flowering. Because the 2 D–H) and pollen and self-pollinated seed fertilities observed in Tve female fertility of (caudata)-Tve was almost normal, as shown different stages of the reconstruction of (caudata )-Ae. caudata above, the near-complete seed sterility of the bagged ears in- polyathera are summarized in Table 3. In this table, the cauda- Tve “ Tv′ ” c′ p′ t′ dicated complete pollen sterility of (caudata)-Tve, which was ta plasmon is abbreviated as c , whereas C ,C ,C , and Dc′ genomes are hybrid genomes derived, respectively, from confirmed by microscopic observation of pollen grains (Fig. 1 B t c t p c t c and C). Thus, the caudata plasmon is characterized as the male C C ,CC ,CC , and DD genomes. The proportion of PMCs sterile plasmon to Tve wheat, as Kihara (15) recognized earlier, showing the modal chromosome configuration is given in pa- and we conclude here that male sterility induced by the caudata rentheses after its configuration. In step 7 of stage II, SB1 plants = = plasmon has stayed unchanged during repeated backcrosses with having 2n 32 and 2n 33 were selected for further backcrossing. normal wheat pollen for 63 generations. Seed setting rates of the crosses and backcrosses, as well as seed germination during the reconstruction, are compiled in Production of germless grains. Another prominent genetic effect of × the caudata plasmon on wheat phenotype was the production of Table 4. Percent cross success was calculated by [% seed set] [% seeds germinated]/100. germless grains by pollination with normal wheat pollen. Their “ Tv′ ” In stage I, we crossed (caudata)-Tve SB50, abbreviated as C , GENETICS occurrence in (caudata)-Tve was discovered in its SB15 genera- ′′ tion (16). This type of mature grain (Fig. 1D, Right) lacked an which showed regular chromosome pairing of 21 at the MI stage (Fig. 2D and Table 3, step 1), with Ae. caudata typica,producing embryo, leaving a cavity that would be filled by the embryo in a Tve t = normal grain (Fig. 1D, Left). These grains never germinated after (caudata )-ABC DF1 plants with 2n 28 chromosomes that showed a modal meiotic chromosome configuration of 1′′′ + 5′′ + soaking. Their occurrence in (caudata)-Tve after pollination with 15′ in their PMCs (Table 3, step 2), in the hope of directly normal Tve pollen was observed in successive backcross gener- transferring the caudataTve plasmon from (caudata)-Tve SB to ations: Table 1 summarizes germless grain frequencies for six 50 Ae. caudata. However, the F hybrids showed complete female groups of SB generations. The overall frequency of germless 1 sterility, setting no seeds in 1,522 florets backcrossed with the Ae. grains produced in the SB –SB generations of the (caudata)- 15 63 caudata pollen (Table 4, step 2); their female sterility appeared to Tve was 11.9%, compared with 0.022% in normal Tve, indicating beduetotheformationoffivebivalentsandonetrivalentinthe a 540-fold increase by the Ae. caudata plasmon. The genetic modal chromosome configuration in the MI stage, which dis- effect of the caudata plasmon in inducing germless grains in Tve turbed unreduced gamete formation that might occur when almost wheat did not change for almost 50 generations of repeated all chromosomes were univalent (Table 3, step 2). Therefore, we backcrosses with normal wheat pollen. tried to produce an alloplasmic octoploid, (caudataTve)-AABBCt Genetic effect of the caudata plasmon on other phenotypic characters of t ’ – C DD, by colchicine treatment of the above F1 plants using Sears Tve wheat. An experiment was conducted in the 1992 1993 crop method (17), and obtained a single chromosome-doubled tiller season to evaluate the genetic effect of the Ae. caudata poly- among four colchicine-treated plants, which set 11 seeds by open athera plasmon on 19 characters of wheat described in Materials pollination (Table 4, step 3); however, crossing between this and Methods. The characters were compared in euplasmic Tve alloplasmic octoploid and Ae. caudata typica as the male was, and alloplasmic Tve SB42 plants (Table 2). again, unsuccessful (Table 4, step 4), even though the female A significant genetic effect of the caudata plasmon was found parent formed bivalents almost regularly in its PMCs (Fig. 2E and only on male fertility, expressed by a great reduction in pollen Table 3, step 4). The cause of this female sterility may have been fertility and two types of self-pollinated seed fertility observed in an unusual genomic ratio between the nuclei involved in double the field and greenhouse. A significant increase in second in- fertilization, namely, four genomes in the egg cell, eight genomes ternode length is probably due to an extreme random fluctuation in the polar nuclei, and one genome in the male gamete, com- pared with their 1:2:1 genome ratio in normal fertilization. In stage II, forced to change our strategy, we used Ae. cylin- Table 1. Frequency of germless grains produced in the SB to 15 drica, a tetraploid species with the CcCcDcDc genome constitu- SB generations of (caudata)-Tve pollinated by normal Tve 63 tion, for bridging the transfer of the caudata plasmon from wheat pollen (caudataTve)-AABBCtCtDD to Ae. caudata: We crossed (cau- SB generations No. of grains No. of Germless dataTve)-AABBCtCtDD as the female to Ae. cylindrica, obtaining Tve t c c grouped observed germless grains grains, % (caudata )-ABC C DD F1 hybrids with a cross success rate of 7.2%, calculated by (cross success rate) × (germination rate) and SB15-SB20 2,113 324 15.3 shown as a percentage (Table 4, step 5). The modal chromosome SB21-SB30 387 57 14.7 configuration in their PMCs was 1′′′′ + 1′′′ + 11′′ + 13′, in- SB31-SB40 151 43 28.5 dicating that most chromosomes in the A and B genomes existed SB -SB 337 48 14.2 41 50 as univalents in the MI stage of PMCs, whereas 14 chromosome SB -SB 1,575 78 5.0 51 60 pairs of the C and D genomes mostly formed bivalents with a few SB -SB 465 50 10.8 61 63 tri- and quadrivalents (Table 3, step 6). These F hybrids were Total: SB –SB 5,028 600 11.9 1 15 63 backcrossed with pollen of Ae. cylindrica (Table 4, step 6) and Control: Normal Tve 4,571 1 0.022 produced SB1 seeds with only a 2.3% cross success. The SB1

Tsunewaki et al. PNAS Latest Articles | 3of9 Downloaded by guest on September 29, 2021 Table 2. Genetic effects of the Ae. caudata polyathera plasmon 41 plants were pollinated with polyathera pollen, yielding hybrid on 19 characters of common wheat strain Tve, extracted seeds in nearly 90% of the pollinated florets (Table 4, step 13). – from the results of a field test carried out in the 1992 1993 The F1 hybrids showed a modal chromosome configuration of 7′′ crop season in more than 90% of PMCs with low pollen but high female Character Unit (ast)-Tve (cdt)-Tve Difference† F value fertility, demonstrating a normal seed setting rate after artificial pollination with Ae. caudata polyathera pollen (Table 4, step 13). WV Grade 0.0 0.0 0.0 0.000 After three successive backcrosses of the hybrid with the polyathera GV Grade 0.0 0.0 0.0 0.000 Tve pollen, the genetically stable SB3 strain of (caudata )-caudata HD Days 30.0 31.0 +1.0 0.772 polyathera was obtained (Fig. 2H and Tables 3 and 4, step 16); thus, PH Centimeter 154.3 147.4 −6.9 6.220 the reconstruction of Ae. caudata polyathera was accomplished, CL Centimeter 143.1 139.3 −3.8 0.688 bringing together its genome and plasmon, which had been sepa- IL1 Centimeter 68.2 65.2 −3.0 1.190 rated from each other for almost 60 generations. Entire spikes of IL2 Centimeter 28.4 29.1 +0.7* 10.343 the initial plasmon and genome donors, and of all genetically stable IL3 Centimeter 16.8 17.9 +1.1 1.007 SB strains produced in the course of Ae. caudata polyathera re- IL4 Centimeter 10.2 9.5 −0.7 0.180 construction, are shown in Fig. 3A, and entire plants of both the − CD Millimeter 5.0 4.8 0.2 0.481 reconstructed Ae. caudata polyathera SB plant and its native plant − 3 SpN Number 36.8 36.1 0.7 7.392 are shown in Fig. 3B. SpL Centimeter 16.8 16.9 +0.1 0.002 StN Number 24.7 24.0 −0.7 2.373 Phenotypic Comparison of Native and Reconstructed Ae. caudata − DW Gram 7.0 6.5 0.5 0.861 polyathera Strains. Experimental design and abbreviations of the PF Percent 94.5 0.9 −93.6 (Not tested) characters investigated are given in Materials and Methods. The data SFF Percent 93.4 0.0 −93.4** 785.490 obtained are shown in SI Appendix; they were subjected to analysis − SFG Percent 87.6 0.0 87.6** 423.960 of variance to detect significant differences between the two strains − CSF Percent 78.5 69.0 9.5 1.495 for all 22 characters, and the results are shown in Fig. 3C as the GR Percent 100.0 100.0 0.0 0.000

CD, culm diameter in the middle of the third culm internode; CL, culm length; GR, germination rate; GV, growth vigor; PF, pollen fertility; SFF, self- pollinated seed fertility of field-grown plants; SFG, self-pollinated seed fer- tility of greenhouse-grown plants; WV, winter variegation. *P < 0.05; **P < 0.01. † [(cdt)-Tve] − [(ast)-Tve].

hybrids thus produced were checked for somatic chromosome number in root-tip cells. We anticipated obtaining a certain number of 2n = 28 plants with a genome constitution of CCDD or, at worst, 2n = 29 plants; the results are shown in Table 5. Surprisingly, we obtained no plants with 2n = 28 or 2n = 29, and we therefore grew 2n = 30–33 SB1 plants. Two with 2n = 30 and 2n = 31 did not grow to heading, so we had to use 2n = 32 and 2n = 33 plants for further backcrossing in stage II, step 7 (Tables 3 and 4). In their SB2 progeny, we obtained three plants that formed 14 bi- valentsin46%oftheirPMCs(Fig.2F and Table 3, step 8), which were used as the female parent in further backcrosses for two more generations until the establishment of a stable strain of Tve c c c c (caudata )-C C D D at the SB4 generation, forming 14′′ in most PMCs [i.e., the stable (caudataTve)-Ae. cylindrica strain] (Tables 3 and 4, step 8′). Tve In stage III, (caudata )-Ae. cylindrica SB2 plants were crossed as the female to Ae. caudata typica (Table 4, step 9), giving rise to Tve c t c F1 seeds of (caudata )-C C D . The hybrids thus produced showed a modal chromosome configuration of 1′′′ + 6′′ + 6′, which was found in about 40% of PMCs (Table 3, step 10) with no seed setting by self-pollination, although they produced seeds in 7.6% of the florets artificially pollinated with Ae. caudata typica Fig. 2. Time course of the transfer of Ae. caudata polyathera plasmon from (caudata)-Tve SB50 to Ae. caudata polyathera.(A) Five stages of the re- pollen (Table 4, step 10). Two successive backcrosses of the F1 construction work, including the prestage of breeding the (caudata)-Tve hybrids and their SB offspring with the same pollen parent pro- 1 SB50 plant. Each black arrow indicates a 1-y single step, except where the duced SB2 progeny that showed normal meiosis, stably forming interval is indicated by red letters. (B–H) Chromosome configurations ob- 7′′ and full fertility in both sexes (Fig. 2G and Tables 3 and 4, served at the MI stage of PMCs of two founder euplasmic parents and five step 12); this strain was (caudataTve)-Ae. caudata typica. genetically fixed strains produced in the course of reconstructing the Ae. At the end of this stage, the senior author (K.T.) found that caudata polyathera strain. (B) Euplasmic Ae. caudata polyathera with 7′′, the Ae. caudata strain used by Kihara (15) as the plasmon donor used as the plasmon donor. (C) Euplasmic T. aestivum strain Tve with 21′′, used as the genome donor in the prestage. (D)(caudata)-Tve SB56, instead of to (caudata)-Tve was Ae. caudata polyathera, and not the variety Tve (caudata)-Tve SB50, with 21′′, produced in the prestage. (E)(caudata )- typica; this point will be explained in Discussion. Thus, we needed t t AABBC C DD at the C4 generation, with 27′′+ 1′, a hypo-octoploid with one to replace the caudata typica genome with that of polyathera, and Tve chromosome missing, produced in stage I. (F)(caudata )-Ae. cylindrica SB3 Tve undertook the experiments of stage IV. First, we crossed (cau- with 14′′,producedinstageII.(G)(caudata )-Ae. caudata typica SB3 with 7′′, Tve Tve data )-caudata typica SB3 plants to Ae. caudata polyathera produced in stage III. (H)(caudata )-Ae. caudata polyathera SB3 with 7′′, KU6-1 as the male on a large scale: A total of 404 florets of produced in stage IV. (Scale bars: B–H,10μm.)

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1817037116 Tsunewaki et al. Downloaded by guest on September 29, 2021 Table 3. Modal chromosome configurations observed at the MI stage of PMCs and pollen and self- or open-pollinated seed fertilities in plants at different stages of the reconstruction of (caudataTve)-Ae. caudata polyathera MI chromosome configuration Selfed seed fertility

No. of florets Stage/step Materials 2n PMCs observed Modal configuration (%) Pollen fertility, % observed Seed set, %

Stage I Tv 1(c )-Tve SB50 42 586 21′′ (97.4) 4.5 652 0.0 Tv t 2(c )-ABC DF1 28 852 1′′′+ 5′′+ 15′ (13.5) — 1,832 0.1 4(cTv)-AABBCtCtDD 55 53 27′′ + 1′ (24.5) — 402 54.6 Stage II Tv t c c 6(c )-ABC C DD F1 42 46 1′′′′ + 1′′′ + 11′′ + 13′ (10.9) — 586 0.0 Tv c c′ c c′ 7(c )-C C D D SB1 32–40 —— —

2n = 32 SB1 32 84 11′′ + 10′ (34.5) — 358 0.9

2n = 33 SB1 33 164 14′′ + 5′ (69.5) — 1,114 2.3 Tv c c c c 8(c )-C C D D SB2 28 796 14′′ (45.6) — 292 65.0 Tv c c c c 8′ (c )-C C D D SB4 28 383 14′′ (86.0) 58.2 274 54.3 Stage III Tv c t c 10 (c )-C C D F1 21 1,144 1′′′ + 6′′ + 6′ (39.9) — 536 0.0 Tv t t′ 11 (c )-C C SB1 14 1,535 7′′ (84.3) 70.8 242 63.2 Tv t t 12 (c )-C C SB2 14 1,945 7′′ (94.2) 92.2 294 85.2 Stage IV Tv t p 13 (c )-C C F1 14 963 7′′ (92.6) 17.9 427 0.9 Tv p p′ 14 (c )-C C SB1 14 1,574 7′′ (99.9) — 121 78.5 Tv p p 15 (c )-C C SB2 14 163 7′′ (100) 92.5 356 67.1 Tv p p 16 (c )-C C SB3 14 —— 97.7 211 96.2 GENETICS

performance of the reconstructed strain relative to that of the WCt3, WCt5, and WCt11, although no difference was detected native strain. between them in 20 WMt loci, as shown in SI Appendix,S2Appendix. We did not find any significant differences (P < 0.05) between This confirmed our mistake in using var. typica, instead of var. pol- the two strains for all 22 characters investigated: thus, we may yathera, in stages I and III of the Ae. caudata reconstruction conclude that the plasmon of (caudataTve)-Ae. caudata polyathera work. SB3 is identical to that of the native polyathera strain, as for their effects on all morphological, physiological, and reproductive Discussion characters investigated. Plasmon Donor to Kihara’s(caudata)-Tve. Ae. caudata consists of two varieties, typica and polyathera. Kihara (15) did not note Analysis of Simple Sequence Repeat Markers of Organellar Genomes. which variety he used as the donor of caudata plasmon in his Comparative analyses of simple sequence repeat (SSR) loci in (caudata)-Tve production. A clue was obtained from records in both organellar genomes were conducted with chromosomally stable 1949, when Kihara made the initial cross between Ae. caudata strains produced in the course of reconstructing (caudataTve)-Ae. and T. aestivum Tve as a pollen parent (15). Referring to the caudata polyathera and its native polyathera strain. Their SSR catalog of Aegilops–Triticum germplasm preserved in Kihara’s band patterns are collectively shown in SI Appendix, S2 Appen- laboratory at Kyoto University (18), his Aegilops–Triticum col- dix, and representative SSR band patterns of two chloroplast lection contained only one Ae. caudata accession at that time, DNA loci, WCt12 and MCt19, and two mitochondrial DNA loci, namely, var. polyathera with the accession number KU6-1, gifted WMt7 and WMt15, are shown in Fig. 3D. by H. Kappert, University of Berlin, Germany, in 1934. From this The euplasmic Ae. caudata polyathera (genome CpCp) and all document, we can conclude that the Ae. caudata accession used five alloplasmic strains having its plasmon but differing in their in Kihara’s 1949 crossing was Ae. caudata polyathera with genome constitution displayed identical band patterns for all accession number KU6-1. This conclusion led us to realize our 22 chloroplast and 20 mitochondrial SSR loci analyzed, whereas mistake in using Ae. caudata typica instagesIandIIIofthere- their patterns differed from those of all of the euplasmic lines construction of Ae. caudata. with different genome constitutions, namely, AABBDD (Tve), CcCcDcDc (Ae. cylindrica), and CtCt (Ae. caudata typica), in three Experimental Proof of Genetic Autonomy of the Plasmon. We dem- to 29 of the 42 loci tested in the two organellar genomes (Table onstrated that the Cp plasmon of Ae. caudata polyathera main- 6). These results showed that the Ae. caudata polyathera plasmon tained its genetic identity unchanged for more than half a has not changed at all, as far as these SSR loci are concerned, century of coexistence with the wheat genome (AABBDD) and during its coexistence with the various foreign genomes de- with three other genomes (AABBCtCtDD, CcCcDcDc, and CtCt) scribed above over 60 generations; this finding critically dem- for shorter periods, regarding its effects on plant phenotype and onstrated the genetic autonomy of the plasmon from the on SSRs in the chloroplast and mitochondrial DNAs. coexisting nuclear genomes. Using the Correns’ repeated backcross method, Michaelis (19) Finally, two other phenomena were noted during this work. produced an alloplasmic line of Epilobium [i.e., (luteum)-hirsu- First, colchicine treatment of plants did not affect the SSR loci in tum SB23 generation] from an F1 hybrid, Epilobium luteum (\) × the organellar genomes, although it caused chromosome dou- Epilobium hirsutum (_), by repeated backcrosses with E. hirsu- bling from 2n = 28 (genome constitution ABCD) to 2n = 56 tum pollen for 23 generations. He made reciprocal crosses, E. (AABBCCDD) in the nucleus. Second, the plasmons of two Ae. luteum (\) × (luteum)-hirsutum SB23 (_) and (luteum)-hirsutum caudata varieties, polyathera and typica, were distinct; they SB23 (\) × E. luteum (_), and found that the hybrids obtained showed electrophoretic differences in three chloroplast SSR loci, by those crosses looked similar to each other and also to the

Tsunewaki et al. PNAS Latest Articles | 5of9 Downloaded by guest on September 29, 2021 Table 4. Seed setting rates of the crosses and backcrosses, and seed germination rates observed in different stages of the reconstruction of (caudataTve)-Ae. caudata polyathera No. of Cross combination No. of ears No. of florets No. of Seed seeds No. of seeds Seeds Cross Stage/step (\ × _) pollinated pollinated seeds set set, % sown germinated germinated, % success, %

Stage I Tv 1(c )-Tve SB50 × 32 758 96 12.7 43 12 27.9 3.5 caudata typica Tv t 2(c )-ABC DF1 × 74 1,522 0 0.0 ——— 0.0 caudata typica 3 Colchicine treat. 57 1,832 11 0.6 4 4 100.0 — Tv t of (c )-ABC DF1 4(cTv)-AABBCtCtDD × 13 294 0 0.0 —— — 0.0 caudata typica Stage II 5(cTv)-AABBCtCtDD × 45 1,002 96 9.6 16 12 75.0 7.2 cylindrica Tv t c c 6(c )-ABC C DD F1 × 117 2,656 86 3.2 83 59 71.1 2.3 cylindrica Tv c c′ c c′ 7(c )-C C D D SB1 × 148 2,796 700 25.0 240 194 80.8 20.2 cylindrica

2n = 32 SB1 × 41 772 200 25.9 120 102 85.0 22.0 cylindrica

2n = 33 SB1 × 101 1,904 488 25.6 120 92 76.7 19.6 cylindrica Tv c c c c 8(c )-C C D D SB2 × 30 398 347 87.2 6 6 100.0 87.2 cylindrica Tv c c c c 8′ (c )-C C D D SB3–4 × 12 198 180 90.9 12 12 100.0 90.9 cylindrica Stage III Tv c c c c 9(c )-C C D D SB2 × 69 954 734 76.9 56 18 32.1 24.7 caudata typica Tv c t c 10 (c )-C C D F1 × 139 974 74 7.6 59 18 30.5 2.3 caudata typica Tv t t′ 11 (c )-C C SB1 × 34 167 138 82.6 70 44 62.9 52.0 caudata typica Tv t t 12 (c )-C C SB2 × 44 281 267 95.0 7 7 100.0 95.0 caudata typica Stage IV Tv t t 13 (c )-C C SB3 × 41 404 357 88.4 24 23 95.8 84.7 caudata polyathera Tv t p 14 (c )-C C F1 × 82 506 173 34.2 70 70 100.0 34.2 caudata polyathera Tv p p′ 15 (c )-C C SB1 × 14 69 64 92.8 20 20 100.0 92.8 caudata polyathera Tv p p 16 (c )-C C SB2 × 18 157 121 77.1 21 14 66.7 51.4 caudata polyathera

E. luteum × E. hirsutum F1 hybrid, all showing normal growth consider how far the plasmon retained its genetic autonomy and pollen fertility. From these observations, he concluded that during polyploid evolution of this complex. Our plasmon classi- “the result was the proof that the cytoplasm retains its specificity, fication is based on the genetic effects of the plasmons of those even when an alien nucleus lies in the cytoplasm for more than species observed for 21 phenotypic characters of 12 common 20 generations. The nucleus cannot change the specificity of the wheats (14, 21) and molecular differences in their chloroplast plasmon” (19). Michaelis’ conclusion of 1965 on plasmon au- and mitochondrial DNAs, which were detected by restriction tonomy was the same as ours of 2018; thus, we confirmed his fragment length polymorphism analyses (23–25). The results conclusion 53 y later, proving, moreover, that the principle of revealed many cases in which a group of polyploid species dif- genetic autonomy of the plasmon holds true for a much longer fering in their genome constitution have an identical or very time scale in the Triticum–Aegilops complex, which seems to be similar plasmon to each other and to a diploid species having a supported by self-replication of organellar DNAs in both the related genome: The first example is a group of three polyploid chloroplast and mitochondrion, as suggested by Allen (20). wheat species, Triticum araraticum, Triticum timopheevii (both 2n = 28, genome AAGG), and Triticum zhukovskyi (2n = 42, Plasmon Autonomy Detected in Polyploid Evolution of the Triticum– AAAAGG), all having a common plasmon “G” of Ae. speltoides Aegilops Complex. The plasmon shows great genetic diversity, with (2n = 14, SS), where the G and S genomes are closely related to 18 plasmon types and five subtypes among 47 strains belonging to each other (26). The second group includes Aegilops triuncialis 31 species of the Triticum–Aegilops complex (14, 21, 22); here, we (2n = 28, UUCC), Aegilops biuncialis, Aegilops columnaris (both

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1817037116 Tsunewaki et al. Downloaded by guest on September 29, 2021 Table 5. Somatic chromosome numbers in lated that those Ae. speltoides accessions are the closest relative of root-tip cells of SB1 plants produced by the B plasmon donor to emmer wheat (22). Similarly, we could not Tve t c c 2 backcrossing (caudata )-ABC C DD F1 hybrids find any diploid species having a plasmon closely related to the D = = (2n 42) with Ae. cylindrica (2n 28) pollen plasmon of Ae. crassa 4x among all diploid species, although the Chromosome D plasmon of Ae. squarrosa was most closely related to the D2 number (2n) No. of plants obtained plasmon in all criteria of the phenotypic effects and restriction fragment patterns of both chloroplast and mitochondrial DNAs 28 0 29 0 among the plasmons of all other diploid species (22). Fig. 4 30 1 summarizes the results of all these considerations. 31 1 32 5 33 15 34 10 35 11 36 8 37 5 38 1 39 2 40 1 41 0 42 0 Total 60

2n = 28, UUMM), and 4x and 6x Aegilops triaristata (2n = 28,

UUMM, and 2n = 42, UUMMNN, respectively), all having the GENETICS plasmon U of Aegilops umbellulata (2n = 14, UU). The third group consists of Aegilops kotschyi and Aegilops variabilis (both 2n = 28, SvSvUU) having the plasmon Sv of Aegilops searsii (2n = 14, SsSs). The fourth group contains Ae. cylindrica (CCDD) and Aegilops ventricosa (DDMM), both having the plasmon D of Aegilops squarrosa (synonym of Aegilops tauschii;2n= 14, DD), and the fifth group contains Ae. triuncialis (2n = UUCC), having the plasmon C of Ae. caudata (2n = 14, CC) (27). Based on these data, we concluded that the above tetraploids originated from hybridization between the diploid species in the same group as the female parent and some other diploid species as the pollen parent. In addition, there are two cases in which one of the tetraploid species thus produced became the female parent of the hexaploid species of the same group through hybridization with its pollen parent or with a third diploid species; such cases are T. zhukovskyi in the first group and Ae. triaristata 6x in the second group, respectively. For all polyploid species of the above five groups, we could trace their plasmon donor back to the diploid species of the respective groups, supporting the proposed plasmon autonomy. Two additional groups consisting of 4x and 6x species having the Fig. 3. (A) Spikes of two founder parents, euplasmic Ae. caudata polyathera same types of plasmon are the emmer wheat and Aegilops crassa used as the plasmon donor to all alloplasmic strains (1) and euplasmic Tve Triticum dicoccoides Tve groups; all species of the emmer group, namely, wheat (2), and of five alloplasmic strains: (caudata)-Tve SB63 (3), (caudata )- t t Tve and many cultivated emmer species, carry AABB genomes and B AABBC C DD colchicine-induced octoploid (4), (caudata )-Ae. cylindrica SB3 Tve Tve plasmon, whereas all hexaploid species, including T. aestivum, pos- (5), (caudata )-Ae. caudata typica SB2 (6), and (caudata )-Ae. caudata sess AABBDD genomes and B plasmon, indicating that the hexa- polyathera SB2 (7). (B) Intact plant of native Ae. caudata polyathera (1) and a ploid common wheat was produced from an emmer wheat(s) as the reconstructed plant from its genome and plasmon that was extracted from (caudata)-Tve SB50 (2). (C) Morphological, physiological, and reproductive female and Ae. squarrosa (genome DD) as the pollen parent. Tve characters of the (caudata )-Ae. caudata polyathera SB3 strain relative to Similarly, three hexaploids, Ae. crassa 6x (DDMMDD), Aegilops those of the native Ae. caudata polyathera strain, taking the latter’s per- vavilovii (DDMMSS), and Aegilops juvenalis (DDMMUU), have formance as 1.0. No characters showed a statistically significant difference at 2 the D plasmon of Ae. casssa 4x (genome DDMM), suggesting P < 0.05 between two strains. (Character names and full performance data that all these 6x species originated from crosses between Ae. are provided in SI Appendix, S1 Appendix). ApslAL, awn length of the apical crassa 4x as the female and different diploid species having a spikelet; FD, flowering date; PS, pollen sterility; N, tiller number; OSF, open- D, S, and U genome, respectively, as their pollen parent; evidently, pollinated seed fertility. (D) SSR electrophoretic band patterns of two chlo- plasmon autonomy has also operated in the evolution of these roplast DNA loci, WCt12 and WCt19, and two mitochondrial DNA loci, WMt7 and WMt15, of euplasmic Tve (1), euplasmic Ae. caudata polyathera KU6-1 (2), two hexaploid groups. Tve Tve (caudata)-Tve SB60 (3), (caudata )-synthetic octoploid C4 (4), (caudata )-Ae. We could not identify any B plasmon donor to the emmer- cylindrica SB (5), (caudataTve)-Ae. caudata typica SB (6), and (caudataTve)-Ae. common wheat group, although the S plasmon found in two of 3 2 caudata polyathera SB2 (7), and molecular markers with lengths in nucleotides the four Ae. speltoides accessions studied was most closely related indicated (M). All five alloplasmic lines having the caudataTve plasmon showed to the B plasmon of this polyploid wheat group. Thus, we postu- the same electrophoretic patterns as Ae. caudata polyathera KU6-1.

Tsunewaki et al. PNAS Latest Articles | 7of9 Downloaded by guest on September 29, 2021 Table 6. Number of total (denominator) and differential (numerator) WCt and WMt loci, detected among four euplasmic and five alloplasmic strains WCt locus WMt locus Total of WCt and WMt loci

Strain 1 2 3 Strain 1 2 3 Strain 1 2 3

2 19/22 —— 2 10/20 — 229/42—— 3 16/22 17/22 — 3 10/20 6/20 — 3 26/42 23/42 — 4 17/22 16/22 3/22 4 10/20 6/20 0/20 4 27/42 22/42 3/42

Strain 1 is T. aestivum (Tve), strain 2 is Ae. cylindrica, and strain 3 is Ae. caudata typica (all three are euplasmic). Strain 4 is a group of six strains consisting of euplasmic Ae. caudata polyathera and five alloplasmics [(caudataTve)-Tve t t SB60, -AABBC C DD, -Ae. cylindrica SB4,-Ae. caudata typica SB2,and-Ae. caudata polyathera SB2].

As described above, plasmon autonomy was recognized in the the middle of the third culm internode; spike number per plant; spike length two-step evolutionary process, 2x → 4x → 6x (Fig. 4, a, 1), in two (SpL); spikelet number per spike (StN); air-dried weight (DW) of a set of three species groups; the S, AG, and AAG genome group, and the U, UC, culms, including seeds; pollen fertility; self-pollinated seed fertility of field- and UM, and UMN genome group, as well as in two one-step evolu- greenhouse-grown plants; artificially cross-pollinated seed fertility (CSF); and seed germination rate. Two plants each of the two strains (euplasmic tionary processes, 2x → 4x (Fig. 4, a, 2)and4x→ 6x (Fig. 4, a, 3), the s s and alloplasmic) were grown in each plot, with four replications, and the data former of which was found in three species groups (S and US ; obtained were subjected to analysis of variance to detect a significant effect of D, CD, and DN; and C and UC genome groups) and the latter in the caudata plasmon on the above characters of Tve wheat, although analysis twospeciesgroups(ABandABDandDM,DMD,DMS,andDMU of variance on the CSF was made using the data of five consecutive years from

genome groups). In the remaining three diploid and polyploid spe- 1989 to 1993 for both (ast)-Tve and (cdt)-Tve, the latter of which was of SB38 to cies groups, no strict plasmon autonomy was detected between the SB42 generations, whereas pollen fertility could not be tested by analysis of diploid and polyploid species, probably due to extinction of their variance, because we could not take any replication in sampling. diploid plasmon donors (Fig. 4, b). In total, seven species groups containing 5 diploid, 10 tetraploid, and 6 hexaploid species displayed Comparative Phenotypic Studies of Native and Reconstructed Ae. caudata plasmon autonomy in their evolutionary processes. This means that polyathera Strains. The phenotypic difference between native and recon- 16 of the 19 polyploid species in the Triticum–Aegilops complex structed Ae. caudata polyathera strains was studied for the following 22 mor- phological, physiological, and developmental characters: HD and flowering maintained plasmon identity through their evolution, where emmer date, both in May; tiller number at maturity; PH; lengths of the first to fifth and common wheat are counted as one species. internodes of a culm from the base to the top of three culms (IL1–IL5);SpL;StN; Contrary to the genetic autonomy observed in many polyploid awn lengths of the first to seventh lateral spikelets (1LslAL–7LslAL) from the species, a large portion of the plasmon diversity had occurred base of each of three spikes in a plant; awn length of the apical spikelet; pollen during diploid speciation: Of the 18 major types and five sub- sterility observed in 300–500 pollen grains per plant; open-pollinated seed types of plasmon found among 47 accessions of this complex, fertility; and DW of the entire plant. The experimental design adopted was a 15 major types and two subtypes were detected among diploid randomized block design, consisting of the above two strains and 14 replica- species. This means that 74% (17 of 23) of the plasmon diversity tions, having a single plant of each strain per plot. The data obtained were occurred at the diploid level. Speciation of diploids is considered subjected to analysis of variance to detect significant differences in these characters between the two strains, among which percent seed fertility was to have occurred in the Pliocene period, 5.3–2.3 My ago (28–30), – – converted to angle before analysis, because the number of seeds obtained from whereas tetraploid wheat originated 0.7 0.4 My ago (29 32), three spikes observed in a plant was much smaller than 100 grains. although no information is available for originated age of other – polyploid species. Diploid species of the Triticum Aegilops SSR Analysis of Organellar DNAs. Seven lines were examined for SSR loci: (i) complex may thus have had sufficient evolutionary time to per- T. aestivum erythrospermum strain Tve, the recurrent pollen parent of (caudata)-Tve mit genetic differentiation of both the nuclear genome and the plasmon, whereas most polyploid species have not yet experi- enced such a long evolutionary time, and have thus retained the original plasmon since their emergence. There is a lot of cross-talk and movement of DNA elements between organelle and nuclear genomes, as well as epigenetic al- terations (33, 34), which contradict the proposed plasmon auton- omy. In general, eukaryotes have a lineage of generative cells, which are called “germ line,” as the ancestral to gamete-forming tissues in the development of an organism (2). In such germ cells, all of the prototypes of the nucleus, plastid, and mitochondrion are assumed to be present to guarantee later development of individuals, and their presence in the germ cell will ensure the plasmon autonomy, although their presence needs to be proved in the future. Materials and Methods Genetic Effect of Ae. caudata Plasmon on Phenotype of Tve Wheat. To de- termine the genetic effect of this plasmon on wheat phenotype, 19 characters other than pollen and seed fertility and germless grain production, which were examined separately, were compared between euplasmic Tve, abbre-

viated as (ast)-Tve, and alloplasmic Tve SB42 plants that were grown in the Fig. 4. Possible evolutionary steps in the Triticum–Aegilops complex, in field in the 1992–1993 crop season. Those characters were winter variega- which genetic autonomy of the plasmon is retained (A) or is suspected (B). tion and growth vigor, both of which were observed in midwinter and Red letters and red arrows show the plasmons that retained genetic au- classified into four grades [from 0 (normal) to 3 (lethal)]; heading date (HD), tonomy in the indicated evolutionary steps, whereas genome (haploid counting April 20 as zero; plant height at maturity (PH); culm length; first to phase) and plasmon types are given for each species using normal and italic fourth internode lengths from the top of the culm (IL1–IL4); culm diameter in letters, respectively.

8of9 | www.pnas.org/cgi/doi/10.1073/pnas.1817037116 Tsunewaki et al. Downloaded by guest on September 29, 2021 SBs; (ii) Ae. caudata polyathera (accession no. KU6-1), the initial plasmon donor were amplified as 80- to 200-bp fragments using primers specific for each

to all five alloplasmic lines produced later; (iii)(caudata)-Tve SB60,instead locus; the three excluded loci did not produce the expected discrete band of its SB50 generation that was used as the secondary source of the cau- patterns. The amplified products were electrophoresed, and the banding pat- Tve data plasmon at the start of the reconstruction work; (iv)(caudata )- terns were visualized using silver staining. Stained products usually appeared as t t Tve c c c c AABBC C DD produced in stage I; (v)(caudata )-C C D D SB2 produced in a cluster of two to five discrete bands, among which the most intensely am- Tve t t Tve stage II; (vi)(caudata )-C C SB3 produced in stage III; and (vii)(caudata )- plified band was used as the representative of the locus of interest. p p C C SB2 produced in stage IV. We also examined euplasmic strains of (viii) Ae. cylindrica and (ix) Ae. caudata typica, which were the genome donors ACKNOWLEDGMENTS. Two Ae. caudata varieties, typica and polyathera, used in stages II and III, respectively. were kind gifts of Shoji Ohta, Fukui Prefectural University. The collaboration Total DNA was extracted from frozen leaves collected in the rosette stage of Tatsuya Yotsumoto in chloroplast and mitochondrial SSR locus analysis is of all strains, according to the method of Liu et al. (35). Previously, Ishii et al. deeply appreciated. This work was carried out at the National Institute of (36, 37) identified 24 and 21 SSR loci, designated WCt1-24 and WMt1-21,in Genetics, Mishima, Faculty of Agriculture, Kyoto University and at Fukui chloroplast and mitochondrial DNAs, respectively, of common wheat cv. Prefectural University, using field and greenhouse facilities managed by Chinese Spring. Of these, WCt7, WCt20, WCt21, and WMt16 are located in Takashi Yoshida (National Institute of Genetics), the late Reiji Matsuo (Fac- coding regions; WCt10, WCt11, WCt12, WMt5, WMt11, and WMt13 are lo- ulty of Agriculture, Kyoto University), and Ken-ichi Oshita (Fukui Prefectural cated in introns; and all others are located in intergenic regions. According University), to all of whom we express our appreciation. We extend our to Ishii et al. (36, 37), these short-repeat loci consist of a simple sequence of appreciation to S. Sakamoto, M. Sugiura, and T. Kuroiwa for providing use- 10–15 of the same nucleotides. All except three, WCt14, WCt17, and WMt19, ful information and for helpful discussions.

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