A Unique Cytoplasmic–Nuclear Interaction in Sunflower (Helianthus annuus L.) Causing Reduced-Vigor Plants and the Genetics of Vigor Restoration

Wild Helianthus species are an important genetic resource for sunflower improvement, but sometimes there are adverse interactions between the wild and cultivated sunflowers. This study reports the inheritance of reduced vigor and its restoration resulting from an interaction of perennial Helianthus cytoplasms with nuclear genes of cultivated sunflower lines. The large number of vigor restoration (V) genes identified in cultivated lines are all located at the same locus, designated V1, suggesting a common origin of these genes. Additional V genes derived from the wild perennial species H. giganteus L. and H. hirsutus Raf. are located at a different locus than V1, designated V2. A major difference between the wild annual Helianthus cytoplasms and perennial cytoplasms is the lack of the vigor-reducing cytoplasms, but surprisingly V genes were observed in wild annual H. annuus L. and H. petiolaris Nutt. which were at the same locus as V1. A common vigor-reducing cytoplasmic effect of the perennial Helianthus species and the existence of a common vigor restoration V gene in most perennial Helianthus species could be explained as a result of vigor selection during Helianthus speciation. V1 was mapped on linkage group (LG) 7 of the sunflower genome, using an F2 population derived from MOL-RV/HA 821. V1 co-segregated with an InDel marker ZVG31, with three single-nucleotide polymorphism (SNP) markers, SFW01024, SFW07230, and SFW00604, located above it on the map at a genetic distance of 0.8 cM, and another SNP marker, SFW08671, below it at a distance of 0.4 cM. The physical distance between the two closest flanking SNP markers corresponds to 0.56 and 1.37 Mb on the HA 412-HO and XRQ assemblies, respectively. The tightly linked markers will help select normal vigor progenies when using perennial Helianthus cytoplasms in a breeding program, which will also provide a basis for studying the mechanism of the cytonuclear interaction, and the speciation of annual and perennial Helianthus species.


INTRODUCTION
Crop wild relatives (CWR) are an important genetic resource for crop improvement to biotic and abiotic stresses in many crops, such as wheat, rice, maize, barley, oat, cotton, and soybean (Yumurtaci, 2015;Mammadov et al., 2018). Cultivated sunflower (Helianthus annuus L., 2n = 2× = 34) is one of the few crops native to the United States. The Helianthus genus is well known for its taxonomic complexity, which includes 53 species (14 annual and 39 perennial) and 19 subspecies (Seiler et al., 2017;Anderson et al., 2019). The annual species are all diploid (including cultivated sunflower), and the perennial species include 26 diploid, three tetraploid, seven hexaploid, and three mixaploid species. As with other CWR, wild Helianthus species represent a large unexploited gene pool with genetic variation for different traits, such as resistance to Sclerotinia, Phomopsis, rust, and downy mildew diseases, and parasitic broomrape (Seiler et al., 2017). They are also a source of new cytoplasmic male sterility (CMS) for sunflower improvement. All the annual Helianthus species, except Helianthus agrestis Pollard, can be hybridized with cultivated sunflower using classical crossing methods (Seiler et al., 2017). However, utilization of the perennial diploid species which represent half of the Helianthus genus, is limited by poor crossability and F 1 sterility in wild × cultivated interspecific hybrids. Development of a two-stage embryo rescue technique and a colchicine treatment of seedlings to double the chromosome number have minimized these problems and made it possible to produce interspecific amphiploids (Amp) (Jan, 1988;Sukno et al., 1999). These amphiploids have proven to be extremely valuable in transferring resistance to Orobanche cumana Wallr. (broomrape) race F, and in the introgression of fertility restoration genes into cultivated sunflower Feng and Jan, 2008;Liu et al., 2013).
Another limitation in the use of the perennial wild species is the existence of adverse cytonuclear interactions. Previously, reducedvigor (RV) plants were observed in backcross progenies of an inbred line HA 89 in the cytoplasms of five perennial Helianthus species (H. mollis Lam., H. maximiliani Schrad., H. grosseserratus Mar., H. divaricatus L., and H. angustifolius L.) (Jan, 1992). The characteristics of RV plants included pale-green leaves, significantly reduced plant height, head diameter, seed weight, percent seed set, net photosynthesis, total leaf chlorophyll, and delayed flowering. The plant vigor reduction effects varied among the different cytoplasms. A cytoplasmic component of these effects has been confirmed by the occurrence of all-normal progenies in crosses of HA 89 with pollen from RV plants. Genetic studies suggested that each of the five species has a single dominant nuclear gene controlling plant vigor restoration (Jan, 1992). In addition, the segregation ratios of normal (N) to RV plants observed in the F 2 progeny of diallel crosses among normal plants with heterozygous or homozygous vigor restoration genes derived from the above five interspecific crosses indicated a common perennial cytoplasmic deficiency in these wild perennial species, and that a common vigor restoration gene could restore the plant vigor (Jan, 1995).
The cytoplasmic genome of plants contains 120-140 genes in the mitochondria and 95-100 genes in the chloroplast. Both chloroplasts and mitochondria require the import of nuclearencoded proteins for organelle biogenesis (Levin, 2003). The results of above reciprocal crosses indicated that the phenotypes of RV plants in the progenies of the five perennial Helianthus species (Jan, 1992) may arise from cytonuclear interactions between the nuclear genome of the annual H. annuus and the cytoplasms of the perennials. Cytonuclear incompatibilities may play a role in establishing reproductive isolation among these species (Burton et al., 2013). The study of the reciprocal F 1 hybrids and backcross families of H. annuus and H. petiolaris in xeric and mesic habitats of the parental species suggested that the parental species' cytoplasms were strongly locally adapted and that cytonuclear interactions significantly affected the fitness and architecture of hybrid plants (Sambatti et al., 2008). Using a target enrichment approach, Stephens et al. (2015) studied phylogeny relationships across 37 diploid Helianthus species/ subspecies with a total of 103 accessions using 170 nuclear genes and the chloroplast sequences. Their phylogeny analysis with nuclear genes supported three major clades including a large annual clade, a southeastern perennial clade, and another clade of primarily large-statured perennials. A rapid radiation and/or high levels of reticulate evolution among perennial Helianthus species was suggested in their study. Later, Lee-Yaw et al. (2019) analyzed the phylogenetic relationships among annual Helianthus species and individuals, using nuclear SNPs and chloroplast genomes sequences. The two perennial species H. nuttallii Torr. & A. Gray and H. maximiliani and one more distantly related genus Phoebanthus grandiflora Torr. & A. Gray were separated in a distinct clade when used as outgroups for the annual Helianthus species. These phylogenetic analyses indicated the clear distinction between the nuclear and chloroplast of annual and perennial Helianthus species (Stephens et al., 2015;Lee-Yaw et al., 2019).
Recently, RV and normal progenies have been observed in nine additional perennial Helianthus species when the wild species were used as maternal parents crossed with the cultivated sunflower inbred lines HA 89  , and H. tuberosus L. Therefore, reduced plant vigor and its restoration are commonly observed in utilizing CWR for sunflower improvement. The objectives of this study were to: 1) further examine reduced plant vigor and vigor restoration (V) genes for the vigor-reducing cytoplasms of wild perennial species with respect to cultivated sunflower, wild annual Helianthus species, and perennial H. giganteus and H. hirsutus; 2) examine the relationships among the V genes in cultivated sunflower, wild perennial and annual Helianthus species, and determine the inheritance of vigor restoration; and 3) map the common V gene from cultivated sunflower to a genetic map using an F 2 population derived from the cross of MOL-RV/HA 821.

Plant Materials
Eight wild perennials plus two wild annual Helianthus species were used in this study. The pedigree of the lines used in the study follow the nomenclature system of Purdy et al. (1968). Briefly, the symbol "/" indicated the primary cross and the backcrosses are indicated by numerals at the "/" symbol and placed on the same side of the symbol as the recurrent parent. Then numerals and the recurrent parent are separated by an asterisk "*". The numerals indicate the number of times the recurrent parent was used, example, (H. giganteus/6*HA89). The "//" symbol indicates a secondary cross. Amphiploids were also used in the pedigree designated as i.e. NMS HA89/H. maximiliani 1631, Amp.
To  (Feng et al., 2015). The pedigrees of the materials used in the study are listed in Table 1.

Progeny Test for Plant Vigor for Progenies Derived From MOL-RV and Cultivated Sunflower
Reduced-vigor progeny of H. mollis/8*HA 89 (MOL-RV) were grown in the greenhouse in 1998 and pollinated with 14 cultivated sunflower lines from diverse genetic backgrounds ( Table 1). Vigor-restored normal (N) F 1 plants were selfpollinated and F 2 progeny evaluated in the greenhouse for plant vigor restoration under normal sunflower growth conditions in 1999. The F 2 segregation ratios of N to RV plants were compared to hypothetical ratios using Chisquare analyses.
Eleven cultivated lines homogeneous or with a high frequency of vigor restoration genes (Jan and Ruso, 2000) were emasculated and pollinated with HA 89. All the F 1 s were self-pollinated to obtain F 2 progenies. For each cross, 40 F 2 progenies were planted in the greenhouse to observe the segregation of N and RV plants.

Half-Diallel Analysis of Vigor Restoration (V) Genes in Restoration Lines
To tentatively test the hypothesis that the V genes in cultivated lines originated from a common source, HA 271, HA 234, VNIIMK 6540, Armavir 3497, Issanka, and HA 821 were included in a half-diallel cross. Testcrosses were made by pollinating CMS RIGX-RV plants with a HA 89 background with 15 F 1 s (Jan and Ruso, 2000). The use of CMS RIGX-RV plants as the female parent assured cross-pollination. The testcross progenies were evaluated in the greenhouse for plant vigor segregation.

Molecular Mapping of the V 1 Gene
An F 2 population including 124 individuals of G99/501-625 derived from MOL-RV/HA 821 was used to map the V 1 gene from HA 821. The N and RV segregation of the F 2 progenies were examined in the greenhouse in 1999, and their genotypes was further confirmed by using F 3 progeny grown in the greenhouse in 2013-2014, with 20-40 progeny seedlings each.
Genomic DNA was extracted according to the protocol of the Qiagen DNAeasy 96 Plant Kit (Qiagen, Valencia, CA, USA). The bulked segregant analysis (BSA) method was used for polymorphism screening (Michelmore et al., 1991). The two parents and the two bulks were used for screening. The two bulks included a homozygous normal bulk (Bulk-N) and a homozygous reduced vigor bulk (Bulk-RV), using equal quantities of DNA from 10 F 2 plants for each bulk. The PCR amplification and genotyping for SSR markers followed Liu et al. (2012). PCR amplification was conducted following Tang et al. (2002) with minor modifications. The 15-µl PCR reaction mixture contained 1× PCR buffer, 2 mM MgCl 2 , 0.2 mM dNTPs, 0.27 µM each of the forward and reverse primers, 40 ng DNA and 1-unit Taq DNA polymerase (Qiagen). PCR amplifications were performed using the "touchdown" profile (Lai et al., 2005;Feng and Jan, 2008) in an MJ Research (Watertown, MA, USA) single or Bio-Rad (Hercules, CA, USA) single or dual 96-well thermal cycler. The PCR products were separated on a 6.5% denaturing polyacrylamide gel after denaturation at 95°C for 5 min, at 60 W for 2.0 h (1× TBE) after pre-run for 1.0 h or on a 6.5% nondenaturing polyacrylamide gel at 60 W for 1.0 h (0.5× TBE), on a CBP Scientific gel electrophoresis system. The gels were analyzed after being stained with GelRed nucleic acid gel stain (Biotium Inc., CA, USA) and scanned with a Typhoon 9410 variable mode imager (Molecular Dynamics Inc., CA, USA).
Bulked segregant analyses (Michelmore et al., 1991) were conducted for polymorphism screening using 550 SSR and expressed sequence tag (EST)-SSR primers on 17 LGs of sunflower, following the method of Liu et al. (2012). An additional 30 SSR/EST-SSR and InDel primers on the candidate LG 7 from 23 maps in the Sunflower CMap Database (http:// sunflower.uga.edu/cgi-bin/cmap/map search) were used for polymorphism detection between the two parents. A total of 58 SSR/EST-SSR and InDel primers from LG 7 were used for polymorphism screening between parents and bulks. In addition, 30 sets of semi-thermal asymmetric reverse PCR (STARP) primers were designed according to the sequences of 30 SNP loci previously mapped on LG 7 (Bowers et al., 2012;Talukder et al., 2014;Hulke et al., 2015) following the method of Long et al. (2017). Each set of STARP primer included two universal priming element-adjustable primers (PEA-primers) and two asymmetrically modified allele-specific primers (AMAS-primers) in combination with one common reverse primer (Long et al., 2017). Polymorphism screening for the SNP markers was conducted among the two parents and Bulk-N. The PCR amplification system, program and product separation were performed as described in Liu et al. (2019). Briefly, the PCR program started with initial denaturation at 94°C for 3 min, followed by six cycles of 2-step touchdown PCR program, in which the Ta/e was decreased by 1°C per cycle starting at 94°C for 20 s and then 55°C for 2 min. PCR was continued with another 34 cycles of 2-step program at 94°C for 20 s and then 62°C for 2 min. The amplification was completed with a 2-min extension at 62°C. The PCR products for STARP markers were separated on a denaturing gel on IR2 4300/4200 DNA Analyzer (LI-COR, Lincoln, NE, USA). The polymorphic markers closely linked to the V 1 gene were used for genotyping the mapping population.
The deviation analyses of the vigor trait and marker loci were compared with the expected Mendelian ratios in the F 2 generation using the Chi-square test. The MAPMAKER/Exp version 3.0b program (Whitehead Institute, Cambridge, MA, USA) (Lander et al., 1987) was used for linkage analysis of the phenotypes and molecular genotypes, with a minimum LOD score of 4.0 and a maximum recombination frequency of 0.30. The Kosambi mapping function was used (Kosambi, 1944), with the "error detection on" command. The linkage map was generated using MapChart 2.3 (Voorrips, 2002).

Physical Location of V 1 on LG 7
The analysis of the physical location of V 1 and linked SNP and other markers on LG 7 was conducted by a BLASTn search against HA412.v1.1.bronze.20141015 on websites of the Sunflower Genome Database (https://sunflowergenome.org/) and the XRQ genome (https://www.heliagene.org/HanXRQ-SUNRISE/) on the INRA Sunflower Bioinformatics Resources (https://www. heliagene.org/) by using the sequences flanking the SNP markers and the sequences of other primers. The order of the linked markers on the genetic map of LG 7 was compared with those of Bowers et al. (2012) and Hulke et al. (2015).

Vigor Restoration of Cultivated Sunflower for Progenies With H. giganteus Cytoplasm
Helianthus giganteus 1934 was pollinated by HA 89 and the F 1 plants were obtained via embryo rescue in 1995. One F 1 plant was male-sterile and backcrossed with HA 89. Segregation of N and RV plants was observed in BC 3 F 1. Three normal BC 4 F 1 plants were pollinated with HA 89 pollen and the BC 5 F 1 plants were evaluated for the segregation of N and RV plants. To study the genes controlling vigor restoration and fertility restoration, a normal-vigor and fertile F 1 plant derived from the cross CMS GIG2//CMS GIG2/(NMS HA 89/H. maximiliani 1631, Amp) was selfed. The F 2 progenies were phenotyped for both vigor and male fertility. Additionally, normal-vigor CMS GIG2 (pedigree: H. giganteus/6*HA89, CMS, Normal) plants were pollinated by HA 821 and testcrossed using HA 89 pollen. Progeny segregation patterns were used to determine the allelism of the vigor restoration genes derived from H. giganteus and in HA 821.

The F 1 and Testcross Progeny Test for V Genes Derived From Different Sources
The F 1 s from crossing RV lines of HA 89 or HA 410 with five perennial Helianthus vigor-reducing cytoplasms of H. grosseserratus (GRO-RV), H. angustifolius (ANG-RV), H. salicifolius (SAL-RV), H. hirsutus (HIR-RV), and H. pauciflorus (PAU-RV) to six homozygous vigor restoration lines (HA 821, RF GIG2-MAX 1631, HIR, ANN PI 649856, ANN Bulk, and PET Bulk) were evaluated for segregation of plant vigor in 2016. Due to the discovery of homozygous V genes in both wild H. annuus (i.e. ANN Bulk and ANN PI 649856) and H. petiolaris (i.e. PET Bulk), partial half-diallel crosses among the six homozygous or heterozygous vigor restoration lines were established in the greenhouse in 2016, including three homozygous lines (HA 821, RF GIG2-MAX 1631, and HIR), and three heterozygous lines from ANN bulk, ANN PI 649856, and PET bulk. Then, six progeny plants from each cross were used to pollinate the CMS RIGX-RV with H. pauciflorus cytoplasm in 2016. Segregation of the N and RV F 2 s and testcross F 1 s was evaluated in the greenhouse in 2017. Progeny segregation of either 1N:1RV or no segregation suggests the same V gene for the two parents, and progeny segregation of either 1N:1RV or 3N:1RV suggests the two parents have different V genes. The data from progenies with segregation of 1N:1RV were not shown.

Vigor Restoration of Cultivated Sunflower for Progenies With H. mollis Cytoplasm
Segregation patterns of the F 1 progenies of the RV plants with H. mollis cytoplasm, MOL-RV, crossed with 14 cultivated sunflower lines in 1998 are shown in Table 2. Typical N and RV seedlings are shown in Figure 1. A high frequency of vigor restoration genes was found in the crosses involving 11 cultivated sunflower lines. The crosses of MOL-RV with HA 821, HA 234, and RHA 271 produced only normal progeny, suggesting the vigor restoration (V) genes in those lines were homozygous. The crosses of MOL-RV with HA 89, RHA 801, and Seneca produced only RV progeny, indicating that these lines did not have dominant vigor restoration genes. In addition, another  inbred line, HA 410, doesn't contain V genes (data not shown). Progeny from crosses with the remaining eight lines had a high frequency of normal plants, suggesting these lines contain V genes, although the V genes may not be homozygous. Since the MOL-RV plants were emasculated over several days, this low frequency of RV progeny could also be the result of accidental self-pollination. The F 2 segregation ratios of N and RV plants of the MOL-RV line crossed with 10 cultivated sunflower lines are shown in Table 3. The segregation ratio of 3 N to 1 RV in nine of the 10 crosses was consistent with a single dominant gene hypothesis for control of vigor restoration. The segregation ratio of the cross MOL-RV/ Armavir 3497 did not fit 3 N to 1 RV ratio (c 2 = 4.800, P = 0.028).
The F 2 plants of MOL-RV/Armavir 3497 were also tested for a segregation ratio of 15:1, which could not exclude two loci controlling the vigor restoration in Armavir 3497 (c 2 = 0.960, P = 0.327). However, the seeds used to produce reduced-vigor plants sometimes have lower germination rate compared to normal-vigor plants, so this maybe the most likely reason for the segregation ratio in this F 2 population not fitting a 3:1 ratio. As a group, the homogeneity test with a probability of 0.241 also supports the single dominant gene hypothesis for vigor restoration.

Half-Diallel Analysis of Vigor Restoration Genes in Cultivated Sunflower
The F 1 hybrids of the 11 lines, including HA 271, HA 234, VNIIMK 6540, Armavir 3497, Issanka, HA 821, RHA 296, Peredovik, Smena, P21, and Hopi Dye, crossed with HA 89 were all N. With over 400 F 2 progeny plants, 40 F 2 progeny plants for each cross, there was not a single RV plant observed (data not shown). This suggested that there is no reduced vigor problem caused by cytonuclear interaction in these cultivated sunflower lines. The F 1 progeny of the half-diallel crosses among six cultivated lines all had normal vigor, i.e., HA 271, HA 234, VNIIMK 6540, Armavir 3497, Issanka, and HA 821. The testcross progenies of the half-diallel crossed F 1 s among HA 271, HA 234, VNIIMK 6540, Armavir 3497, Issanka, and HA 821 onto the RV CMS RIGX were all normal, except for the testcross with VNIIMK 6540/Armavir 3497 F 1 , where a segregation ratio of 1 N to 1 RV plant was observed . The 1 N to 1 RV segregation in the testcross using VNIIMK 6540/ Armavir 3497 pollen could be due to a heterozygous F 1 plant that resulted from a rare heterozygous parent. Therefore, the progeny test results suggested that all these lines possess the same V gene, designated V 1 .

Molecular Mapping of V 1 Gene on LG 7
The F 2 population G99/501-625 derived from MOL-RV/HA 821 was used to map the V 1 gene from HA 821. The 124 individuals in this population included 28 homozygous N, 59 heterozygous N, and 33 RV plants, confirmed by the progeny test, with four individuals not having enough seeds for progeny testing. Chisquare analysis indicated that the homozygous N: heterozygous N:RV phenotypes of the F 2 population fit a 1:2:1 ratio (c 2 = 0.450, P = 0.799), suggesting a single dominant gene controlling the restoration of plant vigor. BSA analysis using 550 SSR and EST-SSR primers from all 17 sunflower LGs among the two parents and the two bulks showed two polymorphic markers, ORS966 and ORS328, on LG 7. Further screening of 30 additional SSR markers on LG 7 identified three polymorphic markers, including two SSR markers, CRT136 and HT520, and one InDel marker ZVG31.
Of the 30 SNP markers tested on the LG 7 map, 18 showed polymorphisms between the two parents and Bulk-N (representative primers shown in Figure 2). The polymorphic SNPs were located at the 15.06-42.56 cM region on the scaffold-based genetic map of LG 7 of Hulke et al. (2015). The screening results showed that some markers were far from the V 1 gene, with the Bulk-N containing the band from the RV parent. Therefore, the markers from the 26.94-35.55 cM region of the scaffold-based genetic map of LG 7 of Hulke et al. (2015) were used as the focal point to add more SNP markers around the V 1 gene. Seven polymorphic PCR-based SNP markers from SFW02370 to SFW04010 were used to genotype the F 2 mapping population, which were all co-dominant. The sequences and the product length of these STARP markers were shown in Table 4. As a result, a linkage map including 12 markers (four SSR, one InDel, and seven SNPs) and the V 1 gene was constructed, covering a genetic distance of 36.7 cM ( Figure 3B). The V 1 gene co-segregated with the marker ZVG31, with three SNP markers, SFW01024, SFW07230, and SFW00604, located above V 1 on the map at a genetic distance of 0.8 cM, and another SNP marker SFW08671, below it at a distance of 0.4 cM.    Figure 3B was 3.2 cM, whereas they co-segregated on Figure 3C.

Physical Location of V 1 on LG 7
The sequences of the SNP markers and other markers closely linked to V 1 were aligned to the reference genome sequences of HA 412-HO and XRQ, respectively ( Table 5). The seven SFW SNP markers above and below the V 1 gene (from SFW02370 to SFW04010) on LG 7 span a 6.7-cM genetic distance on Figure 3C, which corresponds to a physical distance of 5.83 and 2.89 Mb on chromosome 7 of the HA 412-HO and XRQ assemblies, respectively. The order of the markers on the genetic maps is generally consistent with their physical order on the genome assemblies of HA 412-HO and XRQ, respectively, except for the order of the three co-segregated SNP markers SFW01024/ SFW07230/SFW00604, and that of ZVG31 and SFW08671. SFW01024 was located above SFW00604 and SFW07230 on the HA 412-HO assembly, whereas it was below them on the XRQ assembly. The order of ZVG31 and SFW08671 was reversed compared between the HA 412-HO and XRQ assemblies. The V 1 gene was mapped between SNP markers SFW01024/SFW07230/ SFW00604 and SFW08671 on LG 7, spanning a 1.2-cM genetic distance on Figure 3B. The physical distance between the two closest flanking SNP markers corresponds to 0.56 Mb at the 91,735,216-92,294,131 bp region (between SNP markers   (Feng and Jan, 2008). Therefore, the F 2 population derived from a normal-vigor and fertile F 1 plant of cross CMS GIG2//CMS GIG2/(NMS HA 89/ H. maximiliani 1631, Amp) segregated for both vigor and malesterility. This F 2 population included 82 N and 35 RV plants, which was consistent with the 3 N to 1 RV segregation ratio expected with single gene control of vigor restoration (c 2 = 1.507, P = 0.220). Meanwhile, the 79 male-fertile to 31 male-sterile progenies also fit a 3 MF to 1 MS ratio (c 2 = 0.594, P = 0.441), indicating a single gene control of fertility restoration. When the two traits, vigor and fertility restoration, were combined, the resulting 53, 26, 22, and nine plants of normal-vigor male-fertile, normal-vigor male-sterile, reduced vigor male-fertile, and reduced vigor male-sterile plants, respectively, fit a 9:3:3:1 ratio (c 2 = 0.748, P = 0.331), indicating the V and the Rf genes are not linked.
Progeny segregation of 14 normal plants of CMS GIG2/HA 821 crossed with HA 89 is shown in Table 6. Ten populations with segregation ratios of N to RV vigor plants fit the 3 N to 1 RV ratio, and four fit the 1 N to 1 RV ratio. CMS GIG2 was a normal plant, but its vigor restoration gene was heterozygous. The segregation of N and RV plants in all these progenies indicated that the vigor restoration gene derived from H. giganteus 1934 is different from the V 1 gene commonly existing in cultivated lines, designated V 2 here.

Other V Genes Derived From Wild Perennial Helianthus Species
The V genes derived from H. hirsutus and H. giganteus were compared to all other detected V genes. The  Table 7). The results of all normal progeny clearly indicated the common cytonuclear interaction defects in the progeny plant with perennial Helianthus cytoplasms and annual nuclear genomes, and the common function of vigor restoration genes from different sources of HA 821, H. giganteus, H. hirsutus, two H. annuus, and H. petiolaris.
The segregation of N and RV progenies of testcross progenies derived from the partial half-diallel crosses among the six homozygous or heterozygous vigor restoration lines are shown in Table 8. No segregation of plant vigor was observed in the progenies derived from the crosses involving four sources with normal vigor (HA 821, ANN Bulk, ANN PI 649856, and PET   (Table 8).

The Existence of Vigor Restoration (V) Genes in Both Wild Helianthus Species and Cultivated Sunflower
Cytonuclear interactions could act as a source of variation for interspecific hybridization and may drive speciation (Levin, 2003). A study on wheat alloplasmic lines carrying the cytoplasm of Aegilops mutica showed that novel nuclear-cytoplasmic interactions can potentially trigger an epigenetic modification cascade in nuclear genes, which eventually change physiological traits, such as dry matter weight (Soltani et al., 2016). The research on Arabidopsis cytolines (each combining the nuclear genome of a natural variant with the cytoplasmic genomes of a different variant) indicated that genetic variation in organelle genomes could impact three seed physiological traits including dormancy, germination performance, and longevity (Boussardon et al., 2019).
Their results also showed that natural parental accessions had contrasted contributions to the cytonuclear effect on germination phenotype depending whether they provided the nuclear or cytoplasmic genomes. In this study, we also detected a reduction of plant vigor with pale-green leaves and stunted growth in interspecific progenies involving different perennial Helianthus species, but only when using the wild perennial species as the maternal parent and cultivated sunflower as the paternal parent. No reduction of plant vigor was observed in the reciprocal crosses, or in progenies derived from the crosses involving wild annual Helianthus species. Therefore, a common cytoplasmic-nuclear interaction defect commonly exists in alloplasmic lines derived from wild perennial Helianthus species. Since the vigor restoration gene was thought to exist in only the perennial Helianthus species, the high frequency of V in cultivated lines was not expected. Because the cytoplasms of other annual species do not cause adverse interaction with nuclear genes in cultivated sunflower, the CMS PET1 cytoplasm (derived from H. petiolaris, an annual species) (Leclercq, 1969) has been used successfully for hybrid sunflower production for about 50 years. However, if there is ever a need to use perennial Helianthus species cytoplasms, the abundance of V genes in cultivated germplasm lines should not hinder the utilization of perennial species cytoplasms in sunflower breeding programs. Our earlier work only demonstrated that wild annual Helianthus species did not produce RV plants, likely because they didn't have vigor-reducing cytoplasms. For the tested materials in this study, the reduced-vigor trait was only observed in the progenies for the crosses using wild perennial species as the maternal parent, not for the two annual species. The current study has shown that the V genes in wild annual Helianthus species, including H. annuus and H. petiolaris, are the same as the V 1 gene that commonly exists in the cultivated sunflower lines. Future research with diverse sources of annual and perennial species may be necessary to determine whether there is deficiency in their cytoplasms causing reduced vigor and the evolutionary role of V gene for the annual Helianthus species.
Similarly, an explanation for the high frequency of a V gene in cultivated lines without any obvious selective advantage is not clear. Since H. tuberosus has been used extensively in the improvement of cultivated sunflower (Fick and Miller, 1997), it is also possible that the V gene is tightly linked with genes controlling desirable agronomic traits and was simultaneously selected and maintained in those lines. As more sunflower genes are mapped, the prevalence of the V gene in cultivated lines may eventually be more clearly explained.

H. hirsutus PI 547174 and H. giganteus 1934 Had a Different V Gene Than Other Helianthus Species
The crosses involving five perennial Helianthus vigor-reducing cytoplasms with six homozygous vigor restoration lines have assessed the commonality of the cytoplasmic-nuclear interaction defect of RV cytoplasms from different perennial Helianthus species, as well as the vigor restoration genes. The segregation patterns of the progenies of the partial half-diallel crosses among the six homozygous/heterozygous vigor restoration lines indicated that H. hirsutus PI 547174 and H. giganteus 1934 had a different V gene than other Helianthus species. For the convenience of future research, we have designated the vigor restoration gene identified in 1992 (Jan, 1992) and those identified in cultivated lines as V 1 , and the V from H. giganteus and H. hirsutus as V 2 , respectively. Although the two V genes are located on different loci, they both can restore the plant vigor of the progeny containing different cytoplasms, which suggests that they can compensate for a common cytonuclear interaction defect causing reduced plant vigor. The segregation of normal plants in the F 1 and F 2 progeny of MOL-RV/P21 indicated that P21 has the V gene to restore the reduced vigor trait (Tables 2 and 3). P21 has been used to produce several amphiploids for sunflower improvement (Liu et al., 2013; North Dakota State University Foundation Seedstocks (NDSUFS), website: https://www.ag.ndsu.edu/fss/ ndsu-varieties/usda-sunflower-inbred-lines). Therefore, in the study of V genes in sunflower, one needs to avoid adding more V genes by carefully checking their pedigree not involving P21 in amphiploids. The V gene discovered in other sources will need to be compared with the V 1 and V 2 genes for allelism, as well as their effectiveness for restoring other perennial RV cytoplasms. Further study of the RV and its restoration caused by the cytoponuclear gene interaction in multi-species may help to elucidate the speciation of annual and perennial Helianthus species.
The V 1 Gene Was Mapped on LG 7 Using SNP and Other Markers According to BSA screening results between Bulk-N and Bulk-RV using the already mapped SSR/InDel markers, the V 1 gene was mapped to LG 7. However, only five markers were linked to V 1 . With the aim of adding more markers close to V 1 , according to the linkage maps with high-density of SNPs, 30 SNP markers were selected to design PCR-based SNP markers. The markers in a focused region from 26.94 to 35.55 cM on the scaffold-based genetic map of LG 7 of Hulke et al. (2015) were used for further genotyping of the F 2 population. Seven co-dominant SNP markers were successfully added to the map on both sides of V 1 . Using the flanking sequences of the SNP markers and primer sequences of other markers, the V 1 gene has been located onto the physical map of chromosome 7 of the sunflower genome, i.e., a 0.56 Mb region on the HA 412-HO assembly, and a 1.37 Mb region on the XRQ assembly. The tightly linked molecular markers identified in this study will facilitate the markerassisted selection for the lines with vigor restoration genes at the early stages of sunflower breeding, especially utilizing the CWR.
The pattern of cytonuclear interactions is the result of a longterm coevolution between nuclear and organellar genomes under selection pressure, which is essential for the proper function of plant cells (Postel and Touzet, 2020). When using organellar markers to evaluate phylogenetic relationships for characterizing genetic diversity, mitochondrial and chloroplast genes often show markedly different phylogenetic patterns from nuclear markers, which is called "cytonuclear discordance" (Lee-Yaw et al., 2019). Phylogenetic analyses using whole-chloroplast sequence data in combination with over 1000 nuclear SNPs in wild annual Helianthus indicate that cytonuclear discordance is widespread both among species and among individuals within species. Since mitochondria and chloroplasts affect key physiological processes, selection may have played a role in driving patterns of plastid variation (Lee-Yaw et al., 2019). In this study, the existence of V genes to different Helianthus cytoplasms and the vigor restoration ability across the Helianthus species provides another piece of evidence for cytonuclear discordance in Helianthus genus. Since there is clear distinction between the nuclear and chloroplast of annual and perennial Helianthus species (Stephens et al., 2015;Lee-Yaw et al., 2019), the V genes contained in the nuclear genomes of both annual and perennial species suggest that the V genes may have evolved before the speciation of annual and perennial Helianthus species.
Many cytonuclear incompatibilities are caused by plastidnuclear incompatibilities, which has been reported in many flowering plants, such as in Passiflora, Oenothera and Pisum. Such incompatibilities often produce lutescent, chlorosis/ virescence, or variegation, because of a decreased photosynthetic function of plastid complex malfunction in the plants (Greiner et al., 2011;Postel and Touzet, 2020). On the other hand, the incompatibility between mitochondrial and the nuclear genomes will often cause CMS (Postel and Touzet, 2020). As a result, several corresponding nuclear Rf gene have been identified and molecularly mapped in sunflower for different CMS sources, such as Rf 1 for CMS PET-1, Rf 4 for CMS GIG2, and Rf 6 for CMS 514A (Horn et al., 2003;Feng and Jan, 2008;Liu et al., 2013).
In this study, we have mapped the vigor restoration gene V 1 to the LG 7 of HA 412-HO and XRQ sunflower assemblies. With these targeted regions containing the V 1 gene, identification of more molecular markers, such as SNPs or SSR, according to the genomic DNA sequences of the sunflower genome will facilitate fine mapping of the V 1 gene, as well as future map-based cloning of the V 1 gene. Further fine-mapping, detailed analysis of the genes contained in the corresponding regions of the two assemblies, plus using microarray and RNA-Seq (RNA sequencing) techniques (Wang et al., 2009) will help to identify candidate genes for plant vigor restoration. Using other methods such as RT-PCR, gene knock-out, or gene-editing will confirm the function of the gene, and thus will reveal the mechanism for vigor reduction and restoration in sunflower and will help to understand the interaction between the cytoplasm and nuclear genes. Therefore, the inheritance study and molecular mapping of vigor restoration gene in this study will also provide evidence for the speciation of annual and perennial Helianthus species. Finally, the results of these studies will provide the basis for better and more efficient utilization of sunflower CWR in crop improvement.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

AUTHOR CONTRIBUTIONS
C-CJ and ZL conceived and designed the research. ZL, GU, C-CJ performed the experiments, ZL and C-CJ analyzed the data and ZL, C-CJ and GS wrote the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
The project was supported by the USDA-ARS National Sclerotinia Initiative, Grant No. 3060-21220-028-00D, the USDA-ARS CRIS Project No. 3060-21000-043-00D, and the Heilongjiang Postdoctoral Fund of China, Grant No. LBH-Z14190. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity lender, provider, and employer.