1. Introduction
Buckwheat is a nutritional and economically cope adapted to harsh environments, which belongs to the genera
Fagopyrum. It has been widely distributed around the world and already praised as a potential functional food for tea, cookies, noodles and so on. Meanwhile, buckwheat contains high-quality proteins with a high content of essential amino acids; retrograded starch; multiple mineral elements; and abundant secondary metabolic products such as flavonoids, phenolic derivatives, and fagopyrin, which are recognized as the major bioactive components for heath improvement and disease treatment [
1,
2]. At the same time, the protein in buckwheat is gluten-free, and it will process buckwheat and its products as an alternative nutritious food to substitute the gluten grains without causing allergens and digestive issues [
3,
4]. Subsequently, buckwheat contains rare bioactive components such as rutin, quercetin, vitexin, anthocyanidins, and myo-inositol, which play an important role in anti-oxidation metabolism of the human body as scavengers of active oxygen and possess healing effects on some chronic diseases like diabetes [
5], fatty liver [
6], and even cancer [
7]. Additionally, it is should be noticed that buckwheat is the only pseudocereal rich in natural rutin, which process buckwheat became a beneficial source of dietary rutin [
2]. Consequently, buckwheat and its food products have been paid more and more attention to due to its valuable bioactive compounds. Meanwhile, buckwheat has also been treated as a substitute for main food especially in high mountainous areas like the Himalayan region, and it has already become a common food in Southwest China, as well as other regions in East Asia, Europe, and North America [
8]. Southwest China is the original birthplace of buckwheat after morphological, cytology and molecular researches due to its complex geographical environments and variable climatic characteristics [
9,
10,
11]. Before the European scientists began to search for wild buckwheat species in China, there were only three buckwheat species,
F. esculentum,
F. tataricum, and
F. cymosum, which were discovered at the end of the 19th century [
12]. Nowadays, 26 buckwheat species of
Fagopyrum have been confirmed and reported by botanists [
13], most of which are wild buckwheat species, with three identified species
F. hailuogouense,
F. luojishanense and
F. longzhoushanense [
14]. On the other hand, molecular markers-based classifications are reliable in taxonomy and phylogenetic researches, and combined with morphological studies, all buckwheat species in genera
Fagopyrum have been divided into two subdivisions, cymosum group and urophyllum group [
9], and this result has been supported by phylogenetic research in recent years [
15].
However, the phylogenetic relationship between different species in
Fagopyrum still needs to be explored because of the insufficient and non-systematic plant materials and non-specificity molecular markers, which also make the results contradictory in different studies. For example, it was considered that
F. cymosum seems more distantly related to
F. esculentum in morphology and isozymes, but the molecular phylogenetic researches based on chloroplast genomes proved that
F. cymosum has a close relationship with
F. tataricum rather than
F. esculentum [
16]. Subsequently, many researchers have reported the phylogenetic relationship between species used different molecular markers, like
matK/trnK [
15,
17],
FLO/LFY [
18],
rbcL-accD [
19], and so on. To elaborate the taxonomy status and the new species identification, however, some of these researches did not use outgroup species for the phylogenetic analysis [
17,
20], and the results are not comprehensive [
16]. Normally, the phylogenetic relationship investigation based on nuclear genome sequence is different to that constructed by chloroplast genome information, which could suggest hybridization in the urophyllum group of
Fagopyrum [
18]. Some results regarding the phylogenetic relationship among buckwheat species is still contradictory and incongruence. The
F. qiangcai is classified into the urophyllum group according to the fruit characterization, as well as this species should be classified into the cymosum group based on the cotyledons criterion which was proposed by Zhou [
15]. Basically, the details of morphology data from different buckwheat could reflect the differences between species. However, it is still a difficult task to identify the wild buckwheat species or groups only based on morphological characters, because it is hard to find a key character to separate different species clearly, especially the transitional species [
13]. Therefore, it will be very meaningful to the wild buckwheat investigation and the molecular breeding, through fully collecting wild buckwheat resources and using specificity molecular markers like
psbE-psbL and
ndhA intron, which have been reported after comprehensive comparative analysis based on the chloroplast genome of buckwheat [
16] and have not been used in buckwheat phylogenetic research yet. Additionally, the previous research was only based on morphological characteristics and single molecular markers such as
RAPD,
AFLP,
matK and so on, which did not provide enough evidence for the phylogenetic relationship verification [
10,
11,
15,
17,
21]. It is also necessary to verify the potential of the utilization of
psbE-psbL and
ndhA intron in the research of the phylogenetic relationships of buckwheat. The research about
psbE-psbL and
ndhA intron could be treated as the useful extension from chloroplast genome research to phylogenetic analysis. On the other hand, it is still needed to reveal the phylogenetic relationship of the recently identified wild buckwheat species
F. luojishanense and
F. longzhoushanense in
Fagopyrum [
14], especially the taxonomy status of these two species.
In this study, we will explore the phylogenetic relationships and taxonomy status of different buckwheat species in Southwest China through the use of multiple molecular markers including psbE-psbL and ndhA intron combined with morphological analysis results at the same time. After that, the potential of psbE-psbL and ndhA intron in phylogenetic research of buckwheat will be uncovered. Our research will provide richer molecular information that helps clearly distinguish the relationship among different buckwheat species and will make a further evaluation of different plastid DNA barcoding sequences in the molecular characterization of wild species and cultivated accessions of Fagopyrum. Afterwards, we would find more potential and credible genetic markers in buckwheat research.
2. Results
2.1. Analysis Based on Morphological Characteristics of Wild Buckwheat
As the edible part of buckwheat, the morphological characteristics of buckwheat grain are the most important index for the evaluation research of buckwheat. In this way, the fruits of different buckwheat species were observed and compared first. The fruit morphological details were shown in
Figure 1. Based on the morphology of different buckwheat fruits, the differences among buckwheat species are easy to reveal. Subsequently, all the buckwheat in this research were divided into two parts. Species whose achenes length were longer than their perianths, were called the big achene group, and the other buckwheat species whose achenes length were almost equal to that of their perianths, were called the small achene group [
22]. In the big achene group, the cultivated species and its related wild species collected from different locations are clearly distinguished with other buckwheat species, including
F. tataricum and
F. esculentum cv.
T12, as well as
F. tataricum (sichuan)
, F. tataricum (yunnan)
, F. cymosum (sichuan)
, F. cymosum (yunnan) and
F. megaspartanum; meanwhile, two wild buckwheat species could also be separated into this group based on the fruit morphology including
F. qiangcai and
F. callianthum with the average length of seeds being more than 4.5 mm. On the other hand, the small achene group was composed of mostly wild buckwheat species including
F. esculentum ssp.
ancestralis, F. luojishanense; F. jinshaense, F. longzhoushanense, F. rubifolium, F. wenchuanense, F. capillatum, F. pugense, F. urophyllum, F. leptopodum, F. crispatifolium, F. lineare, F. gracilipes, F. gracilipes var.
odontopterum and
F. macrocarpum; the average length of seeds was less than 4.5 mm, and most of them were less than 3.5 mm. The smallest seed in these buckwheat species was
F. jinshaense, the average length of seeds was 2.1 mm and the average width of seeds was 1.5 mm, flow with
F. leptopodum and
F. lineare. All in all, it is also indicated that the relationship revealed in
Fagopyrum is quite limited, which divided buckwheat species into several groups directly only based on morphological evidences, and the taxonomy results of individual wild buckwheat still needs to be described.
After that, the principal components analysis (PCA) was processed to reflect the differences among species in
Fagopyrum, which will reduce the dimensionality of the morphology data from leaf, fruits, chromosome, karyotype, and reproductive patterns. The scatter plot drawn by two component factors (the details were showed in
supplementary material 1) after PCA is illustrated in
Figure 2. Based on the scatter plot, all buckwheat species in this research were separated into two parts. The
F. tataricum, F. tataricum (sichuan)
, F. tataricum (yunnan)
, F. esculentum cv.
T12, F. esculentum ssp.
ancestralis, F. cymosum (sichuan)
, F. cymosum (yunnan)
, F. megaspartanum, and
F. urophyllum clustered together; the other wild buckwheat also clustered together. Subsequently,
F. urophyllum was quite distant to the other wild buckwheat, which means
F. urophyllum has some similarities with
F. tataricum and
F. esculentum ssp.
ancestralis in morphological characteristics, especially leaf, fruits and plant height. Interestingly, they were differing considerably in habit and gross morphology. Additionally, the
F. cymosum (sichuan) and
F. megaspartanum showed a closer relationship than other buckwheat. However, it was still different to the identified buckwheat in one component factor, for example,
F. longzhoushanense, and
F. leptopodum were the same in the horizontal component factor, both of them were 0.82. So it is necessary to evaluate the phylogenetic relationship using molecular markers.
2.2. psbE-psbL and ndhA Intron Are the Promising Molecular Markers in Fagopyrum
The phylogenetic trees constructed by the sequence information of
matK,
rbcL-accD,
trnT-trnL,
psbE-psbL and
ndhA intron based on MP/ML/BI methods are shown in
Figure 3. The primers designed for the phylogenetic analysis were showed in
supplementary Table S1 of supplementary material 2, and the electrophoresis for PCR products for the
ndhA intron could be found in
supplementary Figure S1 of supplementary material 2. The out groups here came from Polygonaceae, Caryophyllaceae, and Chenopodiaceae, respectively, which could provide enough sequences information for phylogenetic analysis in
Fagopyrum. Meanwhile, the phylogenetic trees in
Figure 3 show the results of ML analysis; the trees illustrated are completely coincident with the other trees that were constructed based on MP and BI analysis. Subsequently, all the phylogenetic trees only show branches with bootstrap values more than 50, and the star symbols on the branches of phylogenetic trees represent the support rate which was 100/100/1.0.
From our results, the phylogenetic trees built by different molecular markers showed similar topology structure, and it is also clear that all buckwheat species in this study belong to the same genera. Meanwhile, the outgroup came from different genera divided into two subgroups that colored blue and cyan of the branches, and all species from
Fagopyrum were classified into one big group. Additionally, our results showed that all
Fagopyrum species were separated into two subgroups with high internal resolution, which were colored red and green in
Figure 3 and marked as wild buckwheat and cultivated buckwheat respectively. The cultivated buckwheat consisted of cultivated buckwheat and its related wild species, including
F. tataricum, F. esculentum, F. esculentum ssp.
ancestralis, F. cymosum, and
F. megaspartanum.
On the other hand, the topology structure of phylogenetic trees built by
psbE-psbL (
Figure 3E) and
rbcL-accD (
Figure 3C) were more precise than others, which could reveal the relationship between transitional species with similar morphology clearly, such as the polygenetic relationship among
F. luojishanense,
F. longzhoushanense, F. capillatum, F. crispatifolium, F. gracilipes, F. gracilipes var.
odontopterum,
F. qiangcai, and
F. macrocarpum, which could not be clearly revealed using the other molecular marker. Since the phylogenetic tree only showed the evolutionary branches with bootstrap values higher than 50, it indicated
psbE-psbL could be used to investigate the phylogenetic relationship between the transitional species and morphologically similar species which used
matK and other molecule markers which were hard to reveal. Compared with the other widely used marker
rbcL-accD, the phylogenetic tree built by
ndhA intron was better than that of
rbcL-accD, which could reveal the phylogenetic relationship of
F. tataricum, F. esculentum and
F. cymosum precisely. Additionally, the
trnT-trnL seems to not be good for the phylogenetic study of buckwheat compared with others due to the low bootstrap values.
2.3. psbE-psbL + ndhA Intron and psbE-psbL + matK Could Revealed the Relationship between Species Clearly
From the phylogenetic trees in
Figure 4, which illustrated ML trees coincident with the MP and BI methods, we only display the evolutionary branches with bootstrap values higher than 50, which means the low base substitution among species was ignored. All results showed a clear relationship between buckwheat but with higher bootstrap values than only using signal molecular markers, and the topological structure of phylogenetic trees built by three marks was the best out of all the combinations. The results showed all species in
Fagopyrum clustered together and
Oxyria sinensis and
Rheum palmatum clustered together as the outgroup of
Polygonaceae; meanwhile,
Agrostemma githago and
Salicornia bigelovii were divided into other outgroups that came from other sections. On the other hand, all buckwheat species were classified into two subgroups, a wild buckwheat group and cultivated group. In the cultivated group,
F. cymosum, F. tataricum and
F. esculentum were formed into three subgroups and the bootstrap values of these three subgroups were higher than 93, which was different to the other wild buckwheat group. Meanwhile, the
F. tataricum and its related wild species consisted of one subgroup, as well as
F. esculentum and its wild ancestors
F. esculentum ssp.
ancestralis clustering together, and
F. cymosum clustering with
F. megaspartanum, which demonstrated
F. megaspartanum should be divided into
F. cymosum. Additionally, the
F. cymosum subgroup was closer to
F. tataricum subgroup than the
F. esculentum subgroup with the support rate of the branch being 100/100/1.0. In addition, these results also suggested that
F. cymosum was more closely related to
F. tataricum at the molecular level. At the same time, it was found that the relationship between
F. qiangcai, F. macrocarpum, F. crispatifolium, and
F. gracilipes still needed to be processed.
Further, compared with other combinations based on two molecular markers, the phylogenetic trees built by
ndhA +
psbE-psbL and
matK +
psbE-psbL could reveal the relationship among species better than the other two. Due to the ambiguous topological structure in the cultivated group of the phylogenetic tree based on
rbcL-accD +
psbE-psbL and the unclear relationship between transitional species with similar morphology such as
F. luojishanense, F. longzhoushanense, F. crispatifolium, F. gracilipes, F. gracilipes var.
odontopterum and so on. Subsequently, the phylogenetic tree built by
psbE-psbL +
ndhA and
matK +
psbE-psbL further confirmed the reliability of the relationship between wild buckwheat species and the topological structure between two subgroups in
Fagopyrum. More important is the fact that the topological structures and affinity among buckwheat species were basically the same with that which came from the phylogenetic tree based on three DNA barcodes, which are illustrated in
Figure 4A,D,E.
Finally, all consistent phylogenetic trees constructed by multiple DNA barcodes speared buckwheat species into two big groups that had a high bootstrap value of 100, which also proves that the psbE-psbL and ndhA intron could be used as the ideal molecular markers for the study of the evolutionary relationship among Fagopyrum. Meanwhile, we suggested that the ndhA + psbE-psbL and matK + psbE-psbL could distinguish the relationship between buckwheat species reliably.
2.4. The Phylogenetic Relationship between Species in Fagopyrum
To summarize our results for different molecular markers, we could demonstrate the relationship between species in Fagopyrum. Our results from phylogenic trees based on single and multiple DNA barcodes all indicated the Fagopyrum species should be divided into two groups, a wild buckwheat group and cultivated group, which have similar topology structures and stable bootstrap rates. The wild buckwheat group should consist mostly of wild species including F. urophyllum, F. jinshaense, F. leptopodum, F. gracilipes, F. gracilipes var. odontopterum, F. wenchuanense, F. qiangcai, F. crispatifolium, F. rubifolium, F. callianthum, F. lineare, F. capillatum and F. macrocarpum. Meanwhile, the cultivated group should contain F. tartaricum, F. esculentum, F. cymosum and its related wild species from different locations and also F. megaspartanum which we believe should be treated as F. cymosum.
From our results, we inferred that F. callianthum is in a primitive position to the wild buckwheat group, and it clustered with F. wenchuanense and F. pugense, following with F. urophyllum. Meanwhile, F. lineare, F. leptopodum, and F. jinshaense have a relatively close relationship, as well as F. qiangcai, F. luojishanense, F. longzhousahnense, F. gracilipes var. odontopterum-R, F. crispatifolium, F. gracilipes, F. gracilipes var. odontopterum and F. macrocarpum with a close affinity. On the other hand, the cultivated buckwheat and its related wild species from different locations always gathered together, which reflected a small genetic divergence within cultivated species and its related wild species. Additionally, F. megaspartanum should be classified into F. cymosum, and our results also proved that the F. cymosum was more closely related to F. tataricum at the molecular level.
Further, more importantly, psbE-psbL could distinguish the wild buckwheat from cultivated buckwheat accurately during buckwheat resource investigations, as well as the evolutionary distinction between wild species, especially for wild species with similar morphology that cannot be distinguished clearly only by molecular markers such as matK. All in all, our research indicated that the psbE-psbL could further illustrate the relationship between buckwheat species similar to the ndhA intron.
4. Materials and Methods
4.1. Plant Populations
In this study, 25 buckwheat species were used for the morphological and phylogenetic research, including 2 cultivated species, 21 wild species and 2 variants. The wild buckwheat populations were collected during wild buckwheat investigation from 2015 to 2018 in Southwest China. The detail and collected places of different materials are shown in
Table 1.
4.2. Observation and Analysis of Morphological Characteristics
The two months old seedlings of different species were selected for morphological observation. In this research, we mainly focused on the height, leaf and fruit morphology of buckwheat, including plant height, leaves length, leaf width, seeds length and seeds width, as well as the karyotype and the genetic stability of species which was evaluated by self-sterility or not. The diploid and tetraploid were represented by 2 and 4 respectively, meanwhile, the self-sterility and self-infertility were represented by 1 and 2 when we analyzed these data. All measurement data were designed in completely randomized block design and calculated for three individual plant of each species. Additionally, the Principal Component Analysis (PCA) was processed using IBM SPSS statistics v24 (IBM co., New York, NY, USA) to reflect the differences among different species in Fagopyrum that could reduce the dimensionality of the morphology data as well as the karyotype information and so on. The scatter plot of different buckwheat was drawn using two component factors, which could reflect the differences between buckwheat species preliminarily, and the inter-species relationship in Fagopyrum will also be revealed.
4.3. Genome DNA Isolation and Molecular Barcodes Amplification
We used five different plastid DNA barcodes to demonstrate the phylogenetic relationships in
Fagopyrum. In order to validate the promising molecular markers for the wild resources identification and phylogenetic research in the future, two widely used molecular barcodes
matK [
15] and
rbcL-accD [
18,
19], and three intergenic regions of chloroplast DNA, including
ndhA intron,
trnT-trnL and
psbE-psbL, which came from the results of comparative analysis based on chloroplast genomes [
16], were selected in this research.
The primers designed for PCR amplification are illustrated in
Table S1. Meanwhile, the young leaves of buckwheat from individual seedlings were sampled for total genome DNA isolation using a plant genome extraction kit (TaKaRa co., Beijing, China). The sequences of different molecular barcodes were amplified separately, and the amplification was processed as follows: 95 °C for 4 min, 32 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for 60 s, and the final extension for 8 min at 72 °C. After that, the products were cloned into a pMD19-T (TaKaRa co.) vector and sequenced by ABI 377 DNA Sequencer (Thermo Scientific co., Beijing, China), and the doubtful bases were verified with a third sequencing reaction to avoid errors. The length of the
psbE-psbL was about 1300 bp, the length of the
ndhA-intron was about 1100 bp, and the length of the
trnT-trnL was about 1000 bp, respectively.
4.4. Phylogenetic Analysis
The phylogenetic analysis was performed based on different molecular marker sequences of Fagopyrum, and other taxon of dicotyledonous plants such as outgroup including four species Rheum palmatum, Oxyria sinensis, Agrostemma githago and Salicornia bigelovii came from Polygonaceae, Caryophyllaceae, and Chenopodiaceae, respectively, which could provide more information for the phylogenetic trees constriction, and its nucleotide sequence data were obtained from NCBI.
Subsequently, the sequences of different species were aligned by the CLC-Workbench using the blast program (CLC Bio Qiagen, Hilden, Germany). Individual gap positions were treated as missing data. Meanwhile, the sequences at both ends that came from cloning vectors were deleted. After that, the phylogenetic trees were inferred by three different methods including Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian Inference (BI). Meanwhile, the phylogenetic trees based on the ML method were processed by MEGA 7.022 [
40] as well as the bootstrap replicates were 1000. The phylogenetic trees which were based on the MP method were performed using PAUP v4.0b1023 [
41], and the Heuristic search was set to 1000 random addition sequences. About the phylogenetic trees based on BI method was conducted using MrBayes v3.2.624 [
42], with Markov chain Monte Carlo simulations run twice for 2 million generations independently; the phylogenetic trees were used to construct a majority-rule consensus tree after discarding the first 25% of trees.
Then, indeed, in order to further explore the evolutionary trends of different buckwheat species, which also confirms the potential role of psbE-psbL and ndhA intron in the phylogenetic study in Fagopyrum, five different combination of molecular markers were carried out in the construction of the phylogenetic trees, which combined signal molecular sequences together, including matK + psbE-psbL, matK + ndhA intron, ndhA intron + psbE-psbL, rbcL-accD + psbE-psbL, and matK + ndhA intron + psbE-psbL. It could offer more sequences information to verify the evolutionary relationship among buckwheat, as well as cover two high venation reigns which came from LSC and SSC in the chloroplast genome and two widely used DNA barcodes.