1. Top of page
  2. Abstract
  6. Acknowledgements

Genetic mapping of F2 progeny (n = 225) of hybrids between the Sobano variety of common buckwheat (Fagopyrum esculentum Moench) and the Homo wild accession (F. esculentum var. homotropicum) was carried out using randomly amplified polymorphic DNA (RAPD), sequence-tagged site (STS) and seed protein subunit markers, and three morphological traits. Ten linkage groups were identified, involving 87 RAPD markers, 12 STS markers, four seed protein subunit (PS62/PS59, PS49.8/PS51.4, PS44/PS42.9, and PS39.9/PS37.8) markers, and three morphological alleles controlling homo/long style (H/s), shattering habit (Sht/sht), and acute/obtuse achene ridge (Ac/ac), covering a total of 655.2 cM.

Common buckwheat (Fagopyrum esculentum Moench) is an important traditional crop in China (Lin 1994; Chen 2001) that has high functional value. It is widely cultivated throughout the world. Normally common buckwheat is diploid with eight pairs of chromosomes.

Common buckwheat is a distylous self-infertile species consisting of short-style (thrum) and long-style (pin) plants. Sharma and Boyes (1961) showed that distylous self-infertility is controlled by a single gene (or a group of tightly linked genes) in buckwheat, where thrum-type plants are heterozygotes (Ss) and pin-type plants are recessive homozygotes (ss). The unstable yield of common buckwheat results from its self-infertility. Some self-fertile lines of common buckwheat were developed by means of genetic recombination (Ruszkowski 1980), indicating the foreground of common buckwheat new variety development.

Ohnishi (1998) reported a wild self-fertile type (F. homotropicum Ohnishi) native to southwestern China with a homostylous flower. Some studies have indicated that common buckwheat can be crossed with this wild diploid type (Campbell 1995; Hirose et al. 1995). Woo et al. (1999) showed that self-infertility in common buckwheat and the wild type are controlled by the same gene and suggested that the genotype of F. homotropicum is ShSh recessive to S, but dominant over s. Chen et al. (2004) showed that common buckwheat and the wild self-fertile type can be very easily crossed to produce completely normal fertile hybrids with a normal chromosome pairing configuration in metaphase I of pollen mother cells. They suggested that the wild self-fertile type should be named F. esculentum var. homotropicum (Ohnishi) Chen (here simply designated as Homo, 2n = 2x = 16) instead of a different species.

Although buckwheat has attracted increasing attention, compared to the main food crops of rice, wheat and corn (Grimmer et al. 2007; Zhang et al. 2003; Zhang and Chen 2004; Bindler et al. 2007), there are much fewer reports on DNA molecular markers and genetic mapping of buckwheat.

Some reports on DNA polymorphism in buckwheat have focused on the origins and phylogeny of the genus Fagopyrum (Tsuji and Ohnishi 2000, 2001; Sharma and Jana 2002a, 2002b; Wang et al. 2004). There have been few studies on genetic mapping of common buckwheat as yet. The first linkage map published was based on morphology and allozymes (Ohnishi and Ota 1987). Yasuo et al. (2004) constructed an amplified restriction fragment length polymorphism (AFLP) map using F2 progeny of hybrids between distylous self-infertile common buckwheat (Sobano) and the wild relative homostylous self-fertile F. esculentum var. homotropicum (Homo). A more refined linkage map of common buckwheat would be useful for characterizing the buckwheat genome and for investigating genetic effects on phenotypic traits using quantitative trait locus analysis.

In the present study, randomly amplified polymorphic DNA (RAPD), sequence-tagged sites (STS), some important morphological traits, and seed protein subunits were used for genetic mapping of F2 progeny of self-fertile hybrids between self-infertile Sobano plants with a long style and the wild-type homostylous Homo pure line, which is self-fertile, to construct linkage maps of common buckwheat.


  1. Top of page
  2. Abstract
  6. Acknowledgements

Crossing and planting plan

Two accessions of common buckwheat (F. esculentum) were used: the diploid heterostylous self-incompatible variety Sobano and wild self-fertile accession Homo. The former is developed and suppilied by Suedwestdeutsche Saatzucht, Rastatt, Germany, and it has some properties such as non-shattering, obtuse achene (with arc ridge), and big achene, etc. The latter is originated in Yunnan, China provided by Zeller (Tech. Univ. of Munich, Germany), very much similar to the accession of F. homotropicum Ohnishi (F. esculentum var. homotropicum) reported by Ohnishi (1998), Campbell (1995), and Hirose et al. (1995) except highly crossability and compatibility with common buckwheat under lawful pollination (that is, the long style should be pollinated by the long stamen, and the short style by the short stamen) and it has some relative characters to Sobano, such as homostyle (the long style and long stamen type of flowers), strong shattering, acute achene (with acute angle ridge), and smaller achene, etc. (Chen et al. 2004)

Plants were grown in pots of compost (humus soil). At the beginning of the flowering stage, Sobano plants with a long style as the female parent were crossed with Homo as male parents. Since Homo is homostylous and self-fertile, the hybrid plants are all homostylous and self-fertile and were used to produce F2 seeds under strict isolation. The F2 plants were grown in pots of compost under the same conditions in a growth chamber in the Inst. of Plant Genetics and Breeding, and then used to investigate the inheritance of selected morphological characters, RAPD and STS. To map the genes coding for seed protein subunits, some homostylous F2 plants were used for production of F3 seeds by self-crossing. According to the segregation of seed protein subunits in F3 lines, the genotype of their corresponding F2 plants can be identified.

Inheritance of morphological characters

Selected characters of parental, F1 and F2 plants, including shattering habit (shattering/non-shattering), achene ridge (acute/obtuse), and style (homostyle/long style), were scored for inheritance analysis.

Preparation of genomic DNA samples

Genomic DNA samples were extracted from all F2 plants obtained and some parent plants using cetyltrimethyl ammonium bromide (Jiang 2004; Sharma and Jana 2002a, 2002b) and were examined by electrophoresis on 1.0% agarose gel. The DNA concentration was measured by UV spectrophotometry and then was adjusted to 40 ng µl−1 for polymerase chain reaction (PCR).

RAPD and STS analysis

RAPD and STS PCR amplification reactions were performed on a Rotor-Gene 3000 system. The reaction mixture of 20 µl included 2.0 µl of 10× PCR buffer, 1.5 µl of 25 mM MgCl2, 0.4 µl of 10 mM dNTPs, 1.0 µl of 4 µM primer, 0.2 µl of 2.5 U µl−1 Taq DNA polymerase, and 2.5 µl of 40 ng µl−1 template DNA. The reaction conditions were as follows: (1) pre-denaturation at 94°C for 5 min; (2) 40 cycles of denaturation at 94°C for 1 min, annealing at 36°C for 1 min and extension at 72°C for 2 min; and (3) extension at 72°C for 10 min. Amplified DNA fragments were analyzed by electrophoresis on 1.0% agarose gel and visualized with ethidium bromide staining. The gels were photographed using an UV analyzer and an advanced digital camera.

On the basis of preliminary testing of 105 random primers, 19 RAPD primers (Table 1) were selected for genetic mapping in this study. The selected primers exhibit good polymorphism, clear electrophoretic bands and good repeatability.

Table 1.  RAPD primer sequences, annealing temperature (T), number of maternal (P1) and paternal (P2) polymorphism bands, total number of parental bands (Total), and the ratio of polymorphism bands (Rate). The number of RAPD markers fitting a ratio of 3:1 at the 0.01 level is given in parentheses.
PrimerPrimer sequenceTP1P2TotalRate
Total  81(34)108(41)269(75)70.3%

According to NCBI sequence data and preliminary study for common buckwheat, seven STS primers (Table 2) were designed using Primer Premier 5.0 software. The PCR procedures for STS were the same as for RAPD, except for a different annealing temperature (Table 2).

Table 2.  STS primer sequences, annealing temperature (T), number of maternal (P1) and paternal (P2) polymorphism bands, total number of parental bands (Total), and the ratio of polymorphism bands (Rate). The number of STS markers fitting a ratio of 3:1 at the 0.01 level is given in parentheses. * s gene sequence reported by Yashui and Ohnishi (1998).
PrimerPrimer sequenceGene (no. in NCBI)TP1P2TotalRate
P04F: 5′TGAAGAAGTGGTGGGAGAAY12671748°C4(1)3(0)11(1)63.6%
P08F: 5′ACACCCGCAACACTACCTAY35928653°C3(2)1(1)5(3)80.0%
P14F: 5′GAAGAACGCTGCGAACTGCAB10801940°C5(0)4(3)12(3)75.0%
P15F: 5′ATGAGTTTTATTGGTCCTTGTGAY36195650°C1(0)0(0)1(0)100.0%
P23F: 5′CGGATTTGTTGCCGTTCGTGAY24553655°C3(2)0(0)5(2)60.0%
P34F: 5′TCCCCCTCCTTCCTAAs gene*45°C1(1)0(0)3(1)33.3%
P35F: 5′CCCCTCCTTCCTAs gene*45°C2(0)1(1)10(1)30.0%
Total   19(6)9(5)47(11)59.6%

Preparation of seed protein subunit samples

Seeds of common buckwheat Sobano and Homo, their hybrid seeds and F2 seeds were selected for SDS-PAGE analysis of seed protein subunits. Meanwhile, their random 80 F3 lines and eight random seeds per F3 line were also used for SDS-PAGE analysis. On the basis of a preliminary study and the segregation of four pairs of seed protein subunits (PS62/PS59, PS49.8/PS51.4, PS44/PS42.9 and PS39.9/PS37.8) in an F3 line, the genotype of corresponding F2 plant of the F3 line could be inferred.

The procedure for seed protein sample preparation was as follows. A single achene was ground to a powder and added to 100 µl of extraction solution (50 ml of 55% isopropyl alcohol, 20 ml of 1 mM Tris-HCl, pH 8.0, 30 ml of double-distilled water). The sample was shaken in an ultrasonic shaker for 10 min, treated for 30 min at 60°C in a water bath, and left overnight at 4°C. Then the sample was heated for 15 min in a water bath maintained at 60°C and centrifuged (13 000 rpm) at 4°C for 5 min. The supernatant was transferred into a new tube, mixed with 100 ml of solution C (10 ml of 10% SDS, 20 ml of glycerin, 10 ml of double-distilled water) and heated for 30 min in a water bath maintained at 60°C. Samples were then subjected to SDS-PAGE.


Three gel layers were used for SDS-PAGE analysis of buckwheat seed protein, a concentration layer and two separation layers. The main parameters of the two separation layers were as follows: total concentrate of Acr and Bis (T) = 15.6% and 11.3%, respectively, and Rate of Bis in T (C) = 2.6%, at pH 8.8 and 6.8, respectively, with SDS added to each layer. The electrophoresis conditions and staining procedure of Zeller et al. (2004) and Li et al. (2008) were used.

The molecular weight of each band can be identified by comparison with the molecular markers (116.0, 66.2, 45.0, 35.0, 25.0, 18.4 and 14.4 kDa; Fermentas) according to the formula log Mw= K – bX, where Mw is the molecular weight, X is the migration rate, and K and b are constants.

Data analysis and genetic mapping

All bands for DNA fragments that were reproducibly found on agarose electrophoresis gels were scored as dominant markers. The DNA fragment markers were named according to the primer number and DNA fragment size calculated by comparison to a DNA molecular ladder. A χ2-test was used to compare segregation in the F2 population for each RAPD and STS marker with that expected at the 0.01 level. The RAPD and STS markers with segregation fitting a ratio of 3:1 at the 0.01 level were first used to construct a framework for linkage maps.

All markers were scored with a value of 1 for bands or dominant characters and 0 for others. Linkage analysis and calculation of recombination fractions among DNA fragment markers were performed using JoinMap 3.0 (Bindler et al. 2007; Grimmer et al. 2007). RAPD and STS marker linkage maps were then constructed for a logarithm of the odds threshold of 4.0. Data for three morphological trait markers and four pairs of seed protein subunit markers were integrated with data on RAPD and STS markers, and recombination fractions were calculated.


  1. Top of page
  2. Abstract
  6. Acknowledgements

A total of 225 plants of the F2 progeny of hybrids between Sobano and Homo were produced and used for genetic analysis of morphological characters and RAPD and STS markers. Among them, 80 random plants (F2* population) exhibiting homostyle condition and self-fertility were used for obtaining F3 seeds and SDS-PAGE analysis.

RAPD markers

There were 269 DNA fragment bands produced by 19 RAPD primers in the F2 progeny (Table 1). The average number of DNA fragment bands per RAPD primer was 14.16 (range 5–31). The molecular weights of the DNA fragment bands were mostly in the range 300–1500 bp. Of the 269 DNA fragment bands, 189 bands (70.26%) exhibited polymorphism between the parents and segregated in the F2 population. Among these polymorphic bands, 81 amplified from Sobano, with 34 bands fitting a rate of 3:1 at the 0.01 level. There were 108 bands amplified from Homo, with 41 fitting a rate of 3:1 at the 0.01 level.

STS polymorphism

There were 47 DNA fragment bands in the F2 population generated by seven pairs of STS primers (Table 2). The average number of DNA fragment bands per pair of STS primers was 6.72 with the range from 1 to 12. The molecular weights of these DNA fragment bands were mostly in the range 300–1500 bp. Of these, 28 bands (59.57%) were polymorphic and segregated in the F2 population. Among the 19 bands amplified from Sobano (P1), six fit a ratio of 3:1 at the 0.01 level. Among the nine bands amplified from Homo (P2), five fit a ratio of 3:1 at the 0.01 level.

Genetic mapping of molecular markers

After removing the bands without linkage to each other and the abnormal segregative bands, 99 polymorphic bands produced by 19 RAPD primers and seven pairs of STS primers were used to construct genetic marker linkage maps with JoinMap 3.0 (Fig. 1).


Figure 1. Integrated linkage map of Sobano and Homo based on 87 RAPD, 12STS and four seed protein subunit markers, and three morphological character alleles. H/s, Sh/sht, and Ac/ac are the alleles for style, shattering habit and achene ridge, respectively.

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According to the results presented in Fig. 1, the integrated RAPD and STS linkage map of common buckwheat includes ten linkage groups with 87 RAPD markers and 12 STS markers, and covers a total length of 692.40 cM. The average genetic distance per linkage group in the map is 69.24 cM, with an average distance of 6.99 cM between close markers.

Inheritance of morphological markers

Segregation data for three morphological markers are listed in Table 3. It is clear that homostyle, shattering habit, and acute achene ridge are each controlled by a single dominant gene designated H, Sht and Ac, respectively and that there is a linkage relationship between homo/long style and shattering/non-shattering, with an exchange value of 12.6%.

Table 3.  Segregation of three morphological traits in the F2 population.
Morphological traitsSegregationRatioχ2-value
Homo/long style163:623:10.7837
Acute/obtuse ridge159:663:12.0282
Homo/long style – acute/obtuse achene135:37:41:129:3:3:11.541
Acute/obtuse achene – shattering/non-shattering121:55:33:169:3:3:16.403
Homo/long style – shattering/non-shattering149:33:0:439:3:3:1107.713**

The three pairs of alleles controlling homo/long style, shattering habit, and acute/obtuse achene ridge were successfully integrated into the maps of RAPD and STS markers using JoinMap 3.0 (Fig. 1). Among these, H and Sht genes are tightly linked to each other, with an exchange value of 12.57% on linkage group 1, and the Ac gene is located on linkage group 4. Some molecular markers are in close linkage to these morphological characters. For example, S64-601 is tightly linked to the H/s gene with the cross value of 3.4 and to Sht/sht with the cross value of 9.2 and S52-1006 to the Ac gene with the cross value of 1.6.

Genetic mapping of seed protein subunits

Segregation data for four pairs of seed protein subunits (PS62/PS59, PS49.8/PS51.4, PS44/PS42.9 and PS39.9/PS37.8) in the F2* population are listed in Table 4. It is clear that they are all subject to monogenic inheritance and are controlled by the co-dominant allele and located on different linkage groups. These subunit markers were also integrated with the above marker genetic linkage maps (Fig. 1). It is clear that some of the molecular markers are close to seed protein subunit markers; for example, PS37.8/39.9 is close to S48-729, S73-654, and S52-2708 with a genetic distance of <3.0 cM, PS49.8/51.4 is close to S41-2600 (5.1 cM), and PS44/PS42.9 is linked to the S20-1213 marker at a genetic distance of 5.5 cM.

Table 4.  Segregation of four pairs of protein subunits in 80 F2 progeny (F2*) inferred from F3 lines.
Protein subunitsSegregationRatioχ2P
PS62/PS5925:36:191:2:12.900> 0.05
PS49.8/PS51.427:37:171:2:13.675> 0.05
PS44/PS42.915:42:231:2:12.600> 0.05
PS39.9/PS37.819:45:161:2:11.475> 0.05


  1. Top of page
  2. Abstract
  6. Acknowledgements

Genetic map of common buckwheat

Common buckwheat is diploid (2n = 2x = 16) (Lin 1994; Chen 2001; Chen et al. 2007), indicating eight basic genetic maps. AFLP maps of buckwheat were developed by Yasuo et al. (2004). In the present study, RAPD and STS markers and seed protein subunit markers were first used to construct genetic linkage maps of common buckwheat. A total of 217 polymorphic bands were produced by 19 RAPD primers and 7 STS primers. Among these, 99 RAPD and STS markers were used for ten linkage groups. The extra two linkage groups may be a result of the absence of linked and shared markers. The remaining 118 RAPD and STS markers could not be mapped in this study, possibly because of distorted segregation resulting from the heterozygous maternal Sobano. Chen et al. (2007) reported that the s gene for long style is located on chromosome 4E using common buckwheat trisomic lines, indicating that linkage group 1 containing the s gene in the present study corresponds to chromosome 4E. The linkage maps identified, especially linkage groups 1–5, will provide a basis for genetic research into common buckwheat. The markers amplified by primers (P08, P15, P23) designed as protein gene sequences (AY359286, AY361956 and AY245536) have no linkage with four seed protein subunit genes in this study, indicating different loci.

Morphological and seed protein subunit markers

Three pairs of morphological relative character genes (homostyle/longstyle, H/s; shattering/non-shattering, Sht/sht; acute achene/obtuse achene, Ac/ac) were successfully located on the RAPD and STS maps of common buckwheat. Among them, the H and Sht genes are linked to each other on linkage group 1 at a genetic distance of 12.6 cM. The genetic distance between the H and Sht genes in our RAPD and STS maps of Homo in the present study (12.6 cM) is longer than that in the RAPD map (1.3 cM; Yasuo et al. 2004), which may be due to the different crosses and relevant chromosome structure variation. The allele for the character acute achene ridge was first studied and designated as the Ac gene located on linkage group 4.

Li et al. (2008) reported that there are great variations among different accessions of common buckwheat. Zeller et al. (2004) reported the genetic analysis of two pairs of seed protein subunits (20–25 kDa) and four pairs of alleles for seed protein subunits (35–65 kDa) that have been located on a genetic map for the first time in the present study. The way to the construction of linkage relationship of buckwheat seed protein subunits with other genetic markers was first developed by means of F2* progenies inferred from F3 lines in this study.


  1. Top of page
  2. Abstract
  6. Acknowledgements

We are grateful to Dr. Sun Cheng (School of Biology, Shandong Univ.) for some guidance, to Fan Yan, Li Jianhui, Guo Yuzheng, Wang Tian, Zhang Yizhong, Ren Cuijuan, Wang Aiguo, Sheng Maoyin and Du Mingfeng (Inst. of Plant Genetics and Breeding, Guizhou Normal Univ.) for much assistances, and to the Natural Science Foundation of China (30270852, 30471116), Program for New Century Excellent Talents in University (NCET-2004-0913), Special Project of development of animal and Plant Varieties in Guizhou Province (QianKeYu ZhuanZi [2007]026), and Mega-project of China (2006BAD02B06) for providing funds, and to Prof. Mary Scul-lane for proofing the English.


  1. Top of page
  2. Abstract
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