Faba bean (Vicia faba
L.), one of the most important temperate food crops, is widely grown for human consumption in China, Ethiopia, Egypt and the Andean States of South America and for livestock feed in Europe and Australia [1
]. To date, the average faba bean cultivation area is close to 2.5 million hectares annually, which ranks fourth among cool-season crops [2
]. It has been demonstrated that growing faba bean is the most effective strategy for managing soil fertility through crop rotation, which contributes to sustainable agriculture [3
Faba bean seeds together with other relative beans, have high nutritional values as they are excellent sources of protein, carbohydrates, minerals and fibre [4
]. Nevertheless, faba bean also suffer from both biotic and abiotic factors that constrain their productivity and digestibility. Previous studies have demonstrated that condensed tannins are responsible for low-protein seeds and may decrease feed consumption due to their astringent taste [5
]. Condensed tannins from faba bean may also decrease the efficiency of food utilization [6
]. Although several methods, such as cooking and autoclaving, have been used to remove condensed tannins, these processes may also promote other changes in the seed compounds. Meanwhile, a significant problem in tannin removal is the high cost [8
]. Compared to traditional methods, growing cultivars with low-tannin and zero-tannin content are the most effective, economic and environment-friendly strategy.
A previous study first reported the absence of tannins in the white flowers of faba bean varieties; this served an important role in the in vitro digestibility of nutrients in monogastric animals [9
]. According to Picard [9
], there are two inherited recessive genes, zt-1
, that control the zero-tannin characteristic in faba bean and promote a white flower characteristic in the plant. Genetic studies also discovered that the genes in faba bean that control white-flowered plants actually block anthocyanin synthesis [10
]. Breeders usually use crosses between intergeneric and interspecific plants to improve their characteristics. However, faba bean genotype hybrids carrying different zero tannin genes generally give rise to segregating progenies. Therefore, identifying varieties with zero tannins will be helpful for both choosing appropriate crosses for breeders [12
] and representing a reservoir of genes for tannin-free plants. To date, great progress has been made in developing faba beans with zero tannins. Several markers have been mapped to the zt-1
region. The number of markers is still limited, and more markers are needed to fill the gaps for more efficient marker-assisted selection, further fine mapping and map-based cloning of the gene.
Faba bean, a partially allogamous and genetically isolated plant, tolerates no exchange of genes with any other species, including its close relative Vicia narbonensis
]. The perception is that genetic mapping and marker-assisted selection (MAS) in faba bean faces enormous challenges because of its huge genome size (13,000 Mb) [14
], even though faba bean is diploid and has fewer chromosomes (2n
= 12) than other species in the genus Vicia
Various molecular markers have been widely used in faba bean, especially in genetic diversity and relationships among germplasm collections. For example, amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity in 22 recent faba bean elite cultivars [17
]. Zong et al. [18
] subsequently analysed winter and spring [19
] faba bean accessions worldwide using AFLP markers. Linkage maps of the gene controlling zero tannin in faba bean with SCAR markers developed from linked RAPD markers has been published [20
]. Compared with other molecular markers, simple sequence repeats (SSR) markers are based on the amplification of sequence repetitions. It is a simple and repeatable method that can produce abundant polymorphic fragments. Therefore, SSR markers have the advantage of being a valuable tool for constructing genetic linkage maps and marker-assisted trait selection in faba bean breeding efforts.
As mentioned above, Picard [9
] and Bond [10
] first reported that the seed coat of all white flowered varieties of faba bean was free of tannins. The faba bean germplasm M3290, which is originally from the Mediterranean region, is a tannin-free cultivar with white flowers and the zt-1
]. The variety was collected from the International Center for Agricultural Research in the Dry Areas (ICARDA) Syria [1
]. It was then developed by the Qinghai Academy of Agricultural Science and has been widely used in faba bean breeding programmes in China in the past few years (unpublished data).
The objective of this study was to (1) construct a linkage map of the temporary named gene zt-1 the controls the zero-tannin trait in the M3290 variety and (2) identify closely linked markers that could be useful for marker-assisted selection (MAS) in faba bean and further cloning of the gene.
2. Materials and Methods
2.1. Plant Materials
The Qinghai12 variety has coloured flowers (purple) and high tannin content, while the genotype zt-1
] produces white flowers and has a tannin-free seed coat. M3290 was used as the male parent and Qinghai12 was used as the female parent to develop the population lines. An F2
population with 413 plants and the derived F2:3
families with 8–10 plants each that were derived from the cross between the tannin-free cultivar, M3290, and a condensed tannin line, Qinghai12, were used for mapping the tannin-free gene zt-1
. The parents and populations used in this study were grown in the fields of the Qinghai Agriculture and Forestry Academy. A total of 413 F2
progenies were grown during the 2015 growing season on the experimental farm and all F3
lines with ten or twenty plants each were grown in the next spring in 2016. All the parents and progenies were carefully characterized for their phenotypes by their colour in the field and tested for tannin content in the laboratory during the flowering period. The colours of the offspring were classified as two types, e.g., “white” and “purple”, those with the same flower colour as M3290 were scored as “white” and the remainder with the same flower colour as Qinghai12 were scored as “purple”.
A representative collection of 34 elite faba bean accessions from the major faba bean production regions in China, including ten main spring varieties from Qinghai province, four spring varieties from Gansu province, four Yunnan germplasms, six winter varieties from Sichuan province, seven Jiangsu winter cultivars and three Zhejiang winter cultivars, were used to validate the molecular markers identified to be linked to the zt-1 gene. Two main types in China, the spring and winter faba bean, were both selected in this study to compare different ecotypes of faba bean germplasm. All the representative faba bean samples from different areas were grown on the experimental farm at the Qinghai Agriculture and Forestry Academy.
2.2. Tannin Measurement
To enhance the phenotype accuracy and to confirm the tannin content in the genotypes with different colours, the tannin content was determined in the parents and F2
individuals with different flower colours. The Folin-Donis (F-D) method was used to measure the tannin content with a few modifications [21
2.3. DNA Extraction
After measuring the condensed tannin content, the newly expanded faba bean leaves were used to extract genomic DNA. Genomic DNA was extracted using the DS (Sodium Lauroylsarcosine) protocol [22
]. Tannin-free and condensed tannin bulks were established from 20 free (white flower) and 20 condensed (purple flower) tannin content F2
plants, respectively. Bulked segregant analysis (BSA) [24
] was used to identify whether the markers were linked to the gene controlling the zero-tannin characteristic.
2.4. Marker Analysis
A total of 596 pairs of SSR primers were screened between the two parents and bulks. Among them, 128 SSR markers were referred to in Ma et al. [25
], 236 SSR markers were selected from a linkage map developed by El-Rodeny et al. [26
], and the remaining SSR markers (unpublished data) were kindly provided by the Institute of Crop Science, Chinese Academy of Agricultural Sciences (ICP, CAAS). The ISSR markers used in this study were according to Zietkiewicz et al. [27
]. All the primers used in this study were synthesized by Shanghai Sangon Biological Engineering Technology and Services Company Ltd., Shanghai, China.
SSR reactions were performed in a 20 μL reaction volume containing 1 unit of Taq DNA polymerase (TaKaRa), 2 μL of 10× buffer (50 mmol KCl (TaKaRa), 10 mmol Tris-HCl (TaKaRa, pH 8.3), and 1.5 mmol MgCl2 (TaKaRa), 200 μmol of each dNTP (Roche, Basel, Switzerland), 6 pmol of each primer and 50–100 ng of template DNA. The PCR conditions were as follows: denaturation at 94 °C for 4 min; 35 cycles of 94 °C for 1 min, 50–61 °C (depending on primers) for 1 min, and 72 °C for 1 min; and a final extension at 72 °C for 10 min. ISSR-PCR amplifications were performed in 25 μL reaction volumes with 80 ng of genomic template DNA, 2 μL of 10 mM Tris–HCl, 50 mM KCl, 15 mM MgCl2, 0.2 mM of each dNTP, 120 nM of each primer, and 1 U of Taq DNA polymerase. ISSR-PCR reactions were performed with the following conditions: denaturation at 95 °C for 5 min; 35 cycles of 95 °C for 30 s, annealing at optimal temperature for 1 min, and 72 °C for 1 min; and a final elongation step at 72 °C for 10 min.
PCR reactions were performed in a PTC200 Peltier Thermal Cycler. PCR products were then mixed with 4 μL of the formamide loading buffer (98% formamide, 10 mM EDTA, 0.25% bromophenol blue, and 0.25% xylene cyanol, pH 8.0, Shanghai Sangon) and heated at 94 °C for 5 min. The PCR products were separated on 6% denaturing polyacrylamide gels, 8% non-denaturing polyacrylamide gels or 1.5% agarose gels. Each 5–7 μL sample was loaded and then resolved using the silver staining method as described by Bassam et al. [28
] or ethidium bromide and then photographed.
2.5. Statistical Analysis and Genetic Mapping
The Chi-square tests (χ2
) were used to determine the theoretical expectation based on the assumption of a single Mendelian gene controlling zt-1
. Recombination fractions were converted to centiMorgans (cM) and the genetic distances of closely linked markers were calculated with software JOINMAP version 4.0 using the Kosambi mapping function [29
]. A LOD score of 3.0 was used as a threshold for grouping and a maximum recombination fraction of 0.5 were employed as linkage criteria to establish the linkage group [30