Research Article |
Corresponding author: Chuanliang Deng ( dengchuanliang@htu.edu.cn ) Academic editor: Luiz Gustavo Souza
© 2021 Jian Zhou, Shaojing Wang, Li'ang Yu, Ning Li, Shufen Li, Yulan Zhang, Ruiyun Qin, Wujun Gao, Chuanliang Deng.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Zhou J, Wang S, Yu L, Li N, Li S, Zhang Y, Qin R, Gao W, Deng C (2021) Cloning and physical localization of male-biased repetitive DNA sequences in Spinacia oleracea (Amaranthaceae). Comparative Cytogenetics 15(2): 101-118. https://doi.org/10.3897/CompCytogen.v15.i2.63061
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Spinach (Spinacia oleracea Linnaeus, 1753) is an ideal material for studying molecular mechanisms of early-stage sex chromosome evolution in dioecious plants. Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) technique facilitates the retrotransposon-relevant studies by enriching specific repetitive DNA sequences from a micro-dissected single chromosome. We conducted genomic subtractive hybridization to screen sex-biased DNA sequences by using the DOP-PCR amplification products of micro-dissected spinach Y chromosome. The screening yielded 55 male-biased DNA sequences with 30 576 bp in length, of which, 32 DNA sequences (12 049 bp) contained repeat DNA sequences, including LTR/Copia, LTR/Gypsy, simple repeats, and DNA/CMC-EnSpm. Among these repetitive DNA sequences, four DNA sequences that contained a fragment of Ty3-gypsy retrotransposons (SP73, SP75, SP76, and SP77) were selected as fluorescence probes to hybridization on male and female spinach karyotypes. Fluorescence in situ hybridization (FISH) signals of SP73 and SP75 were captured mostly on the centromeres and their surrounding area for each homolog. Hybridization signals primarily appeared near the putative centromeres for each homologous chromosome pair by using SP76 and SP77 probes for FISH, and sporadic signals existed on the long arms. Results can be served as a basis to study the function of repetitive DNA sequences in sex chromosome evolution in spinach.
FISH, Genomic subtraction hybridization, Retrotransposon, Sex chromosome evolution, Spinach
Sex chromosomes evolved from autosomes by stages; the key event in the evolution of sex chromosomes includes the emergence of sex-determining genes, recombination suppression, accumulation of repetitive sequences, degeneration of Y chromosome, and dosage compensation effect of X chromosome (
Repetitive DNA sequences, primarily transposons, retrotransposons (RTs), and tandem repeats (satellite DNA, small satellite DNA, and microsatellite DNA sequences), make up the majority of all the nuclear DNA in most eukaryotic genomes (
Genomic subtraction is used for isolating DNA that is absent in deletion mutants. The method removes the sequences present in the wild-type (tester DNA) and the deletion mutant genomes (driver DNA) from wild-type DNA featured by simple, rapid, sensitive, and economic means. This technique is widely applied in the separation and identification of gene rearrangement and in the preparation of polymorphism loci probe (Straus et al. 1990; Hou et al. 1995;
In this study, the X and Y chromosome of spinach were successfully isolated and amplified by degenerated-oligonucleotide-primed polymerase chain reaction (DOP-PCR) (
The seeds of spinach (S. oleracea Linnaeus, 1753, cv. Japan) were planted in the garden field of Henan Normal University under natural conditions. Genomic DNA from each male and female spinach was extracted from young leaves using the traditional cetyltrimethylammonium bromide method (Rogers et al. 1989).
The X/Y chromosome is the largest submetacentric chromosome (Ellis et al. 1960;
The modified DOP-PCR primer that contains BamH I digestion site (modified primer sequence: CGGAGGATCCNNNNNNATGTGG), was used to amplify the products from the second round spinach Y chromosome DOP-PCR amplification. DOP-PCR amplification was performed in 50 μL reaction volume containing 1 × PCR buffer, 1.5 mmol/L dNTP Mixture (Transgene, Beijing, China), 2.5 U Taq polymerase (Takara, Bejing, China), 100 ng template DNA, and 0.2 μM primer. The amplification was performed by initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing temperature 55 °C for 90 s, extension at 72 °C for 3 min, and a final extension at 72 °C for 10 min. Then, the concentration of the amplified DOP-PCR products was quantified, followed by enzyme digestion in 25.0 μL volume containing 2 × K buffer and 20 000 U BamH I (Takara, Beijing, China) at 37 °C for 3 h in a metal bath for further quantification and quality control.
As described from Y chromosome amplification, the modified DOP-PCR primer was used to amplify the second round DOP-PCR amplification products of spinach X chromosome along with the quality control and quantification of the products. Finally, the amplified products from both X and Y chromosomes were purified using Takara MiniBEST DNA Fragment Purification Kit Ver.4.0 (Takara, Beijing, China).
The DOP-PCR amplified products from the libraries of X (Driver DNA) and Y (Tester DNA) chromosomes were mixed in a 100:1 ratio for subsequent hybridizations. The mixed DOP-PCR amplified products were treated by water bath at 99 °C for 10 min, mixed with 4 mL of PERT (8% phenol, 1.25 M sodium perchlorate, and 0.12 M disodium hydrogen phosphate dissolved in 1000 mL of distilled water) for 72 h, annealed at 25 °C and at 100 rpm on the shaking table for 72 h (shaking for 8 h and stopping for 8 h), and then placed on the shaking table overnight. After 72 h of annealing, the hybridization solution was purified by suction filtration with a syringe and an organic filter. It was extracted twice with chloroform:isoamyl alcohol at 24:1, centrifuged at 12 000 rpm for 5 min, precipitated by 1% volume of sodium acetate and 2.5 volumes of absolute ethanol at -20 °C overnight, centrifuged at 10 000 rpm for 10 min, washed twice with 70% absolute ethanol, dissolved in 800 μL sterile ddH2O, and transferred into a 1.5 mL centrifuge tube. Then, the mass of the hybridization solution was quantified by microspectrophotometer for further steps.
Enzyme digestion, purification, dephosphorylation, and re-purification of the vector were conducted.
A mixture of 1.0 μL of PUC119, 2.0 μL of 10 × K buffer, 2.0 μL of BamH I, and 15 μL ddH2O were quantified into 20.0 μL for 3 h digestion in a 30 °C water bath. Then, purification was performed according to Takara MiniBEST DNA Fragment Purification Kit Ver. 4.0 (Takara, Beijing, China). The reaction mixture was placed in a 0.2 mL centrifuge tube containing 40.0 μL (1–20 pmol) vector DNA, 5.0 μL 10 × K alkaline phosphatase buffer, and 1.0 μL CIAP, and adjusted to 50 μL. The reaction was conducted in a metal bath at 37 °C for 15 min, and then at 50 °C for 15 min for dephosphorylation, purification, and mass quantification.
Gradient design was carried out according to the ratio between the hybrid liquid and the vector, after which the optimized reaction mixture was placed in the microcentrifuge tube containing 1.5 μL PUC119 (Takara Code: 3319), 0.1 µL T4 DNA ligase (Takara Code: 2011A), and 2.0 µL 10 × buffer at contents up to 20 µL. The reaction was conducted in the metal bath at 16 °C for 5 h. The ligation products were transformed into competent cells, screened according to blue and white spots, and amplified by colony PCR using universal primer M13 (CGCCAGGGTTTTCCCAGTCACGAC).
The selected recombinant plasmids were identified using the spinach female and male genomic DNAs as probes labeled with DIG (Roche: 11277065910) by dot hybridization method. Basically, the subtractive DNA libraries with male-specific DNA sequences were hybridized and formed colonies on films. These colonies were selected for further Sanger sequencing.
On the basis of the results of dot-blot hybridization, the male-hybridized colonies (PCR-amplified products derived from bacterial solution with more than 250 bp) were selected for Sanger sequencing at Shanghai Invitrogen Biotechnology Co., Ltd. The sequencing results were analyzed by BLASTn and RepeatMasker (http://www.repeatmasker.org/). Initially, sequencing products were blasted against the spinach reference genome (http://www.spinachbase.org/cgi-bin/spinach/index.cgi) with a cutoff of 90% similarity and E-value 1e-10 to prevent the contamination of the DNA from other organisms. Sequences with no hits were deleted. Then, the DNA sequences were aligned to RepeatMask libraries to classify the type of repeats. Ultimately, the DNA sequences were annotated using BLASTn against the NCBI nucleotide database. Based on the sequencing results, primers for each group of repetitive DNA sequences were designed by Oligo7 for PCR amplification (Suppl. material
The spinach seeds were initially soaked in a moisturized and low-temperature (4 °C) environment overnight. Then, the seeds were placed in a constant temperature incubator at 25 °C in the dark. The seeds with approximate 1 cm root length were placed in a 1.5 mL centrifugal tube for nitrous oxide pretreatment. Subsequently, the roots were fixed in 90% glacial acetic acid for 10 min and finally stored in the refrigerator at -20 °C in 70% ethanol. Each selected tissue was rinsed by distilled water for 10 min, after which it underwent dissection and digestion using a solution containing 1% pectolyase Y23 (Yakult Pharmaceutical, Tokyo) and 2% cellulose Onozuka R-10 (Yakult Pharmaceutical, Tokyo) for 1.5 h at 37 °C (one section per tube with 20 µL of the enzyme solution). The abovementioned treated root sections were carefully split into individual cells by using needles and by intensive vortexing at room temperature along with soaking in 100% ethanol. Furthermore, the cells were collected from the bottom of the tube by centrifugation and re-suspended in an acetic acid ethanol solution (9:1 dilution). Finally, the cell suspension was dropped onto glass slides in a box lined with wet paper towels for observation.
Then, 45S rDNA (the probe was donated by Fangpu Han, a researcher from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) was labeled using Alexa fluor-488-dUTP (green), and the male-specific bands were labeled using Texas-red-dCTP (red) with the nick translation method based on previous protocols (
Fluorescence in situ hybridization (FISH) between spinach chromosomes at metaphase and DNA probes derived from each repetitive sequence was performed according to the method described in previous studies (
The amplified products from the X/Y chromosome were identified using a male-specific marker T11A (
Gel electrophoresis results of spinach Y (A) /X (B) chromosome DOP-PCR products using modified primer Lane M: Trans 2K plus DNA Marker; Lane 1–8(A): spinach Y chromosome second round DOP-PCR products as template using modified primer; Lane 1–8(B): spinach X chromosome second round DOP-PCR products as template using modified primer
From the subtractive library, 2700 single colonies were obtained, among which 480 single colonies with lengths between 250 and 1500 bp were randomly selected for PCR amplification (Suppl. material
Sanger sequencing of the 55 selected DNA sequences yielded a total of 30, 576 bp of product, ranging from 248 bp to 1, 354 bp in length (MN830920–MN830942, MN810356–MN810387). A total of 12, 049 bp of DNA products were identified as repeat sequences using RepeatMasker softerware (http://www.repeatmasker.org/), which accounted for 39.4% of the total sequences. Thirty-two of the 55 DNA sequences contained repeat DNA sequences, including LTR/Copia, LTR/Gypsy, simple repeats and DNA/CMC-EnSpm (Suppl. material
ID | Accession number | Size | Description | Query Cover | E Value | Per. Ident |
---|---|---|---|---|---|---|
SP1-3 | MN830920 | 661 | PREDICTED: Spinacia oleracea uncharacterized LOC110790287 (LOC110790287), transcript variant X4, ncRNA | 96% | 0 | 94.25% |
SP1-71 | MN830921 | 326 | Select seq AP017640.1 Spinacia oleracea DNA, BAC clone: 009-126-13E-1, strain: 03-009, complete sequence | 96% | 3.00E-162 | 100.00% |
SP1-86 | MN810356 | 293 | Select seq AP017641.1 Spinacia oleracea DNA, BAC clone: 009-160-1L-1, strain: 03-009, complete sequence | 93% | 2.00E-103 | 92.03% |
SP1-89 | MN810357 | 294 | Select seq AP017637.1 Spinacia oleracea DNA, BAC clone: 009-26-14K-1, strain: 03-009, complete sequence | 93% | 2.00E-137 | 99.28% |
SP2-26 | MN810358 | 248 | PREDICTED: Spinacia oleracea uncharacterized LOC110800978 (LOC110800978), mRNA | 94% | 1.00E-109 | 97.87% |
SP3-4 | MN810359 | 270 | Select seq XM_021992625.1 PREDICTED: Spinacia oleracea uncharacterized LOC110787992 (LOC110787992), mRNA | 98% | 1.00E-79 | 87.41% |
SP3-8 | MN810360 | 261 | Spinacia oleracea mitochondrion, complete genome | 96% | 3.00E-116 | 97.23% |
SP3-36 | MN830922 | 334 | PREDICTED: Spinacia oleracea uncharacterized LOC110799754 (LOC110799754), mRNA | 96% | 1.00E-160 | 99.07% |
SP3-88 | MN830923 | 293 | Spinacia oleracea DNA, BAC clone: 009-26-14K-1, strain: 03-009, complete sequence | 68% | 3.00E-67 | 91.04% |
SP4-1 | MN810361 | 433 | Select seq XM_022007996.1 PREDICTED: Spinacia oleracea uncharacterized LOC110802566 (LOC110802566), mRNA | 99% | 6.00E-168 | 91.88% |
SP4-2 | MN810362 | 432 | PREDICTED: Spinacia oleracea uncharacterized LOC110791229 (LOC110791229), mRNA | 99% | 0 | 95.82% |
SP4-3 | MN810363 | 423 | PREDICTED: Spinacia oleracea uncharacterized LOC110802566 (LOC110802566), mRNA | 96% | 5.00E-179 | 94.51% |
SP4-4 | MN810364 | 426 | Select seq XM_022001236.1 PREDICTED: Spinacia oleracea uncharacterized LOC110796204 (LOC110796204), mRNA | 98% | 0 | 94.41% |
SP4-7 | MN810365 | 432 | PREDICTED: Spinacia oleracea uncharacterized LOC110802566 (LOC110802566), mRNA | 98% | 0 | 96.49% |
SP4-8 | MN810366 | 433 | PREDICTED: Spinacia oleracea uncharacterized LOC110802566 (LOC110802566), mRNA | 98% | 0 | 96.73% |
SP4-10 | MN810367 | 432 | Select seq XM_022007996.1 PREDICTED: Spinacia oleracea uncharacterized LOC110802566 (LOC110802566), mRNA | 98% | 0 | 96.02% |
SP4-11 | MN810368 | 423 | PREDICTED: Spinacia oleracea uncharacterized LOC110802566 (LOC110802566), mRNA | 98% | 8.00E-177 | 93.68% |
SP4-38 | MN810369 | 293 | Spinacia oleracea DNA, BAC clone: 009-123-11N-1, strain: 03-009, complete sequence | 97% | 1.00E-135 | 97.90% |
SP4-48 | MN830924 | 575 | Select seq AP017641.1 Spinacia oleracea DNA, BAC clone: 009-160-1L-1, strain: 03-009, complete sequence | 36% | 2.00E-61 | 88.78% |
SP4-53 | MN830925 | 262 | PREDICTED: Spinacia oleracea probable methyltransferase PMT15 (LOC110782476), mRNA | 31% | 2.00E-29 | 97.59% |
SP5-1 | MN810370 | 410 | Select seq AP017639.1 Spinacia oleracea DNA, BAC clone: 009-123-11N-1, strain: 03-009, complete sequence | 49% | 2.00E-87 | 96.53% |
SP5-2 | MN830926 | 365 | Select seq AP017641.1 Spinacia oleracea DNA, BAC clone: 009-160-1L-1, strain: 03-009, complete sequence | 56% | 1.00E-94 | 97.61% |
SP5-3 | MN830927 | 365 | Spinacia oleracea DNA, BAC clone: 009-160-1L-1, strain: 03-009, complete sequence | 57% | 2.00E-93 | 97.14% |
SP5-4 | MN830928 | 356 | Select seq AP017639.1 Spinacia oleracea DNA, BAC clone: 009-123-11N-1, strain: 03-009, complete sequence | 56% | 4.00E-55 | 87.13% |
SP5-5 | MN830929 | 534 | Spinacia oleracea DNA, BAC clone: 009-126-13E-1, strain: 03-009, complete sequence | 37% | 7.00E-88 | 97.46% |
SP5-9 | MN830930 | 430 | Select seq XM_022005381.1 PREDICTED: Spinacia oleracea uncharacterized LOC110800092 (LOC110800092), mRNA | 51% | 9.00E-92 | 95.05% |
SP5-10 | MN810371 | 409 | Spinacia oleracea DNA, BAC clone: 009-123-11N-1, strain: 03-009, complete sequence | 50% | 3.00E-86 | 95.63% |
SP5-11 | MN810372 | 485 | Spinacia oleracea DNA, BAC clone: 009-126-13E-1, strain: 03-009, complete sequence | 42% | 4.00E-90 | 97.97% |
SP5-12 | MN830931 | 326 | Select seq AP017640.1 Spinacia oleracea DNA, BAC clone: 009-126-13E-1, strain: 03-009, complete sequence | 96% | 1.00E-161 | 100.00% |
SP5-48 | MN830932 | 277 | Select seq AP017638.1 Spinacia oleracea DNA, BAC clone: 009-41-10L-1, strain: 03-009, complete sequence | 94% | 1.00E-75 | 87.17% |
SP6-20 | MN810373 | 573 | Chain A, Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions | 92% | 6.00E-171 | 87.52% |
SP7-3 | MN810374 | 549 | Select seq XM_021994667.1 PREDICTED: Spinacia oleracea uncharacterized LOC110789945 (LOC110789945), mRNA | 79% | 6.00E-133 | 86.73% |
SP7-4 | MN830933 | 504 | PREDICTED: Spinacia oleracea transcription factor MYB80 (LOC110782202), mRNA | 95% | 0 | 98.96% |
SP7-5 | MN830934 | 587 | Spinacia oleracea mitochondrion, complete genome | 94% | 0 | 98.74% |
SP7-7 | MN810375 | 536 | PREDICTED: Spinacia oleracea uncharacterized LOC110777888 (LOC110777888), mRNA | 53% | 3.00E-116 | 93.73% |
SP7-9 | MN830935 | 504 | Select seq XM_021986329.1 PREDICTED: Spinacia oleracea transcription factor MYB80 (LOC110782202), mRNA | 93% | 0 | 98.94% |
SP7-10 | MN810376 | 536 | PREDICTED: Spinacia oleracea uncharacterized LOC110783205 (LOC110783205), mRNA | 53% | 9.00E-107 | 91.64% |
SP7-11 | MN830936 | 563 | Select seq XM_021982478.1 PREDICTED: Spinacia oleracea pentatricopeptide repeat-containing protein At5g02860 (LOC110777897), mRNA | 94% | 0 | 99.06% |
SP10-9 | MN810377 | 790 | Select seq XM_022003128.1 PREDICTED: Spinacia oleracea uncharacterized LOC110797998 (LOC110797998), mRNA | 98% | 0 | 84.22% |
SP13-1 | MN810378 | 267 | Select seq XM_021992625.1 PREDICTED: Spinacia oleracea uncharacterized LOC110787992 (LOC110787992), mRNA | 97% | 2.00E-83 | 88.97% |
SP13-2 | MN810379 | 267 | PREDICTED: Spinacia oleracea uncharacterized LOC110787992 (LOC110787992), mRNA | 97% | 2.00E-73 | 86.69% |
SP17-1 | MN810380 | 715 | PREDICTED: Spinacia oleracea uncharacterized LOC110799950 (LOC110799950), mRNA | 91% | 0 | 91.10% |
SP17-2 | MN830937 | 1128 | Select seq AP017639.1 Spinacia oleracea DNA, BAC clone: 009-123-11N-1, strain: 03-009, complete sequence | 98% | 0 | 93.99% |
SP51-1 | MN830938 | 758 | PREDICTED: Spinacia oleracea tudor domain-containing protein 3 (LOC110774971), transcript variant X2, mRNA | 51% | 6.00E-178 | 99.71% |
SP51-2 | MN830939 | 766 | PREDICTED: Spinacia oleracea tudor domain-containing protein 3 (LOC110774971), transcript variant X2, mRNA | 45% | 1.00E-179 | 100.00% |
SP51-3 | MN830940 | 758 | PREDICTED: Spinacia oleracea tudor domain-containing protein 3 (LOC110774971), transcript variant X2, mRNA | 51% | 1.00E-179 | 100.00% |
SP52-1 | MN830941 | 644 | Select seq AP017637.1 Spinacia oleracea DNA, BAC clone: 009-26-14K-1, strain: 03-009, complete sequence | 42% | 5.00E-79 | 87.73% |
SP52-3 | MN830942 | 638 | Spinacia oleracea DNA, BAC clone: 009-26-14K-1, strain: 03-009, complete sequence | 42% | 5.00E-69 | 85.87% |
SP55-1 | MN810381 | 1001 | Select seq AP017640.1 Spinacia oleracea DNA, BAC clone: 009-126-13E-1, strain: 03-009, complete sequence | 93% | 0 | 84.31% |
SP55-3 | MN810382 | 1001 | PREDICTED: Spinacia oleracea uncharacterized LOC110802605 (LOC110802605), mRNA | 50% | 0 | 99.28% |
SP55-4 | MN810383 | 1001 | PREDICTED: Spinacia oleracea uncharacterized LOC110802605 (LOC110802605), mRNA | 53% | 0 | 99.78% |
SP73 | MN810384 | 1318 | Select seq AP017638.1 Spinacia oleracea DNA, BAC clone: 009-41-10L-1, strain: 03-009, complete sequence | 100% | 0 | 99.85% |
SP75 | MN810385 | 1354 | Spinacia oleracea DNA, BAC clone: 009-41-10L-1, strain: 03-009, complete sequence | 100% | 0 | 99.93% |
SP76 | MN810386 | 1163 | PREDICTED: Spinacia oleracea uncharacterized LOC110779482 (LOC110779482), partial mRNA | 45% | 3.00E-152 | 85.66% |
sp77 | MN810387 | 1154 | PREDICTED: Spinacia oleracea uncharacterized LOC110779482 (LOC110779482), partial mRNA | 45% | 3.00E-147 | 85.23% |
Using the 32 DNA sequences that contained repeat DNA sequences as probes, we tried to identify the distribution of fluorescence signals on the Y chromosome. However, no fluorescence signals were found on the chromosomes using four simple repeats (SP5-1, SP55-1, SP55-3 and SP55-4) and two DNA/CMC-EnSpm DNA sequences (SP55-3 and SP55-4). When four LTR/Copia DNA sequences (SP3-4, SP3-8, SP17-1 and SP1-86) were selected to be used as probes, the signals showed a dispersed distribution in all chromosomes (Suppl. material
Four pairs of primers were generated according to the DNA sequences SP73, SP75, SP76, and SP77 (Suppl. material
For chromosomal localization, 45S rDNA was used as a probe to distinguish each chromosome, the prominent fluorescent signals of which were observed on chromosomes 2, 5, and 6 (
Distribution patterns of hybridization signals from female and male spinach using 45S rDNA (green) and SP73 (red) as probes A (A’), DAPI B (B’), 45S rDNA (green) as probe C (C’), SP73 (red) as probe D (D’), The merged figure of A (A’), B (B’) and C (C’). Scale bas: 10 μm.
Distribution patterns of hybridization signals from female and male spinach using 45S rDNA (green) and SP75 (red) as probes A (A’), DAPI B (B’), 45S rDNA (green) as probe C (C’), SP75 (red) as probe D (D’), The merged figure of A (A’), B (B’) and C (C’). Scale bars: 10 μm.
Distribution patterns of hybridization signals from female and male spinach using 45S rDNA (green) and SP76 (red) as probes A (A’), DAPI B (B’), 45S rDNA (green) as probe C (C’), SP76 (red) as probe D (D’), The merged figure of A (A’), B (B’) and C (C’). Scale bars: 10 μm.
Chromosome microdissection technology has the advantage of being able to isolate specific DNA products from a single chromosome. Moreover, the isolated products of the target sequences can be enriched through PCR (Zhou et al. 2007). In this study, we combined the conventional genomic subtraction hybridization with single chromosome microdissection to rapidly clone male-biased DNA sequences from spinach sex chromosomes. Twenty-one of 55 cloned DNA sequences were partially overlapped to BAC clone 009-126-13E-1, BAC clone 009-160-1L-1, BAC clone 009-26-14K-1, BAC clone 009-123-11N-1, and BAC clone 009-41-10L-1 located on the male-determining region of the spinach Y chromosome (
Sex reversal from hermaphroditism to dioecy in flowering plants requires two mutants, namely, one male-sterile mutant (generally for the first time) and one female-sterile mutant. These mutant sites are used to stabilize sex on a pair of chromosomes (
This work was financially supported by grants from the National Natural Science foundation of China (31770346, 31970240, and 31470334).
JZ, SW, and CD designed the experiments. JZ and SW conducted the study, processed the data and wrote the manuscript. LY, JZ, NL, SL, YZ, RQ, WG, and CD discussed the results and revised the manuscript. All authors have read and approved the final manuscript.
Figures S1–S6, Table S1
Data type: Figure/Table
Explanation note: Figure S1. Procedure of isolation of biggest chromosome in spinach by micromanipulator. Figure S2. Partial PCR products of recombinant clones using M13R and M13F as primers. Figure S3. 3 Dot blot hybridization results of partial subtractive hybridization clones. Figure S4. Distribution patterns of hybridization signals from female and male spinach using 45S rDNA (green) and SP1-86 (red) as probes. Figure S5. PCR amplification result of SP73, SP75, SP76 and SP77 marker. Figure S6. Pair-wise alignment between SP73 and BAC clone 009-41-10L-1. Figure S7. Pair-wise alignment between SP75 and BAC clone 009-41-10L-1. Table S1. Primer sequences for repetitive DNA sequences. Table S2. Summary of repetitive elements in 55 DNA Sequences.