Construction of a molecular genetic map for hawthorn based on SRAP markers

Abstract

The F1 hybrid population was a result from the crossing of ‘Damianqiu’ and ‘Qiujinxing’. ‘Damianqiu’ was the female parent and ‘Qiujinxing’ was the male parent. Ninety-two F1 individual plants and their parents, in total of 94 materials, were chosen for the sequence-related amplified polymorphism analysis. The Joinmap3.0 software was used to construct the molecular genetic map of ‘Damianqiu’ and ‘Qiujinxing’, respectively. According to the result, the molecular linkage map of ‘Damianqiu’ has 21 linkage groups with 96 markers, which covers a genetic distance of 686.67 cM, with an average interval of 7.15 cM. ‘Qiujinxing’ has 18 linkage groups with 89 markers, which covers a genetic distance of 529.58 cM, with an average interval of 5.95 cM. The aim of this study was to create the first hawthorn molecular genetic map, which could be used as a foundation for gene mapping and assisted selection in the future.

Keywords:

• hawthorn
• SRAP markers
• genetic map

Introduction

Life science is focused on the molecular gene mapping because of its guidance in breeding and genetic engineering. High-density genetic linkage map is the prerequisite and basis for gene mapping and marker-assisted selection (MAS), due to the character of high-genetic heterozygosity, large tree volume, self-incompatibility and long generation cycle. It is very difficult to construct the ideal mapping population (like with the annual field crops), which limits the construction of genetic mapping of fruit trees. But since Hemmat et al. [1] proposed the concept of ‘double pseudo-testcross’ and applied it on apple genetic mapping for the first time, the construction of molecular genetic map of fruit trees has made considerable progress. In recent years, molecular genetic maps have been successfully constructed for most fruit trees, such as apple,[2–4] pear,[5] tangerine,[6] cherry,[7] banana,[8] olive,[9] papaya,[10] peach,[11] lychee[12] and longan.[13]

Hawthorn (Crataegus pinnatifida Bge.) belongs to Rosaceae (Rosaceae) and is native to China. It is an important plant which can be used to make food and medicine. Most of the cultivars and wild species of hawthorn are diploid (2n = 2x = 34). The ‘double pseudo-testcross’, like in other woody fruit trees, could be used for the construction of a genetic map for hawthorn. Since there is no molecular genetic map for hawthorn at present, in this paper we created a hybrid population based on ‘Damianqiu × Qiujinxing’ and constructed the first hawthorn molecular genetic map. This map lays the foundation for quantitative trait locus (QTL) mapping of hawthorn's important traits and marker-assisted breeding.

Materials and methods

Plant material

The mapping population consisted of 92 F1 individual plants crossed by ‘Damianqiu’ (female) × ‘Qiujinxing’ (male) which were planted in 2006. ‘Damianqiu’ as a main cultivar has the character of orange-red peel, large fruit weighing up to 18–22 g, tastes sweet and sour, soft meat, high edible rate, stable yield and diploid; ‘Qiujinxing’ as a high-quality cultivar has the character of strongcold resistance, dark red peel, medium size, tastes sweet and sour, thick aroma, dense and meticulous flesh, shelf-stable, and it's the diploid varieties too. In this study, 92 individual plants from the hybrid group and their parents in a total of 94 materials were used for molecular marker analysis and mapping construction.

DNA extraction

In May 2013 young leaves of hawthorn were picked and the total genomic DNA was extracted using the hexadecyltrimethy ammonium bromide (CTAB) method.

SRAP reaction system and amplification protocol

A stable system of sequence-related amplified polymorphism (SRAP) has been established.[14] The reaction volume was 20 μL, including 10 × PCR buffer (Mg²⁺ free) 2.0 μL, Mg²⁺ 2.5 mmol L⁻¹, dNTPs 0.15 mmol L⁻¹, primer 0.25 μmol L⁻¹, template DNA 20 ng and Taq DNA polymerase 0.5U. The protocol for PCR amplification was initial denaturation (5 min at 94°); denaturation (60s at 94°), annealing (60s at 35°), extension (90 s at 72°), for five cycles; denaturation (60 s at 94°), annealing (60 s at 50°), extension (90 s at 72°), for 35 cycles; final extension (10 min at 72°). The amplification products were separated by electrophoresis on 6% polyacrylamide gels. The gels were visualized by the method of silver staining.

Date acquisition and processing and the map construction

According to the results of electrophoresis, using the number 1 and 0 to represent the marker data, and according to the data, using the ‘CP mapping model’ in the software JoinMap3.0, the LOD D 4.0, maximum recombination rate 0.4, the separated sites of parents were analysed, respectively, we obtained the molecular marker linkage group of the parents. And then Mapchart2.2 was used to construct the linkage map.

Results and discussion

Primer selection

A total of 192 pairs of SRAP primer combinations were used for selection, and 120 pairs of SRAP primers were obtained all of which have a stable amplification, clear banding pattern and good general effect (Figure 1). Forty-seven pairs of primers were used for PCR amplification in large groups (Tables 1 and 2), after electrophoresis, recording the segregation of the markers.

Figure 1. The result of some polymorphism SRAP primers in parents.

Table 1. SRAP primers used in this study.

Forward primers	Reverse primers
me1 5′-TGAGTCCAAACCGGAGC-3′	em1 5′-GACTGCGTACGAATTAAT-3′
me2 5′-TGAGTCCAAACCGGATA-3′	em2 5′-GACTGCGTACGAATTAGC-3′
me3 5′-TGAGTCCAAACCGGAAG-3′	em3 5′-GACTGCGTACGAATTGCA-3′
me4 5′-TGAGTCCAAACCGGACC-3′	em4 5′-GACTGCGTACGAATTACG-3′
me5 5′-TGAGTCCAAACCGGAAT-3′	em5 5′-GACTGCGTACGAATTAAC-3′
me6 5′-TGAGTCCAAACCGGTGT-3′	em6 5′-GACTGCGTACGAATTGAC-3′
me7 5′-TGAGTCCAAACCGGTTG-3′	em7 5′-GACTGCGTACGAATTATG-3′
me8 5′-TGAGTCCAAACCGGTAG-3′	em8 5′-GACTGCGTACGAATTTGC-3′
me9 5′-TGAGTCCAAACCGGTCA-3′	em9 5′-GACTGCGTACGAATTTGA-3′
me10 5′-TGAGTCCAAACCGGAGG-3′	em10 5′-GACTGCGTACGAATTTAG-3′
me11 5′-TGAGTCCAAACCGGAGA-3′	em11 5′-GACTGCGTACGAATTTCG-3′
me12 5′-TGAGTCCAAACCGGAAA-3′	em12 5′-GACTGCGTACGAATTGTC-3′
me13 5′-TGAGTCCAAACCGGAAC-3′	em13 5′-GACTGCGTACGAATTGGT-3′
me14 5′-TGAGTCCAAACCGGGTA-3′	em14 5′-GACTGCGTACGAATTCAG-3′
me15 5′-TGAGTCCAAACCGGACG-3′	em15 5′-GACTGCGTACGAATTCTG-3′
me16 5′-TGAGTCCAAACCGGGGT-3′	em16 5′-GACTGCGTACGAATTCGG-3′
me17 5′-TGAGTCCAAACCGGCAT-3′	em17 5′-GACTGCGTACGAATTCCA-3′
me18 5′-TGAGTCCAAACCGGGAC-3′	em18 5′-GACTGCGTACGAATTGAT-3′
me19 5′-TGAGTCCAAACCGGACA-3′	em19 5′-GACTGCGTACGAATTCTC-3′
me20 5′-TGAGTCCAAACCGGACT-3′	em20 5′-GACTGCGTACGAATTCAT-3′
me21 5′-TGAGTCCAAACCGGCAG-3′	em21 5′-GACTGCGTACGAATTCTA-3′
me22 5′-TGAGTCCAAACCGGCTA-3′	em22 5′-GACTGCGTACGAATTCAA-3′
	em23 5′-GACTGCGTACGAATTCAC-3′
	em24 5′-GACTGCGTACGAATTCTT-3′

Table 2. The combination message of the primers used in this study.

Primer combination	Primer combination	Primer combination	Primer combination
em5me5	em18me20	em8me17	em3me16
em1me8	em19me20	em13me17	em7me16
em2me8	em15me20	em11me17	em9me16
em4m422	em10me21	em16me17	em14me16
em5me22	em5me21	em15me17	em17me16
em6me22	em6me21	em19me17	em5me19
em7me19	em3me21	em17me17	em10me17
em12me19	em2me21	em21me17	em22me20
em13me19	em14me21	em23me17	em6me19
em15me19	em19me19	em23me18	em8me18
em17me19	em3me18	em20me18	em21me20
em20me20	em5me18	em18me18

The ‘e’ indicates ‘em’ and the ‘m’ indicates ‘me’.

The polymorphic markers of the parents and the segregation distortion of the markers

According to the result (Figure 2), we obtained 315 polymorphism markers in the parents, as the number of markers for the male parent is 84 and 55 for the female parent. The result of χ² test (α = 0.01) shows that there were 22 markers that deviated from the Mendelian segregation in the male parent, and accounting for 26.19% of the male parent markers; there were 17 markers that deviated from Mendelian segregation in the female parent, and accounting for 30.91% of the female parent markers. In addition, 176 segregating markers, which belong to both parents (segregating in the population, theoretically segregating ratio 3:1), were also obtained. According to χ² text (α = 0.01), there were 53 marker deviations from the Mendelian segregation, accounting for 30.12% of all the markers. In this study, segregation distortion is evident in all kinds of markers. This degree of segregation distortion has a close relationship with the distance of the parents' kinship; the farther the kinship, the larger the degree of segregation distortion.[16]

Figure. 2. Amplification results of primer e4+m22.

Construction of the molecular genetic map

Using the software JoinMap 3.0, LOD = 4.0, maximum recombination rate 0.4, specific segregation loci of the female parent and the segregation locus which belong to both parents were used for the operation, and the same was done for the male parent. Recombination rate was converted to map distance (cM) by the Kosambi function. Using the Mapchart 2.2 [17] software to construct the molecular genetic map (Figures 3 and 4), we obtained the following result: the molecular linkage map of ‘Damianqiu’ has 21 linkage groups with 96 markers, which covers a genetic distance of 686.67 cM, with an average interval of 7.15 cM. The average length of the linkage groups is 32.70 cM. The average number of loci in per group was 4.57. Among the 21 linkage groups, ‘DMQ1’ has the most loci (35). Two linkage groups have the fewest loci (only 2). ‘DMQ1’ also has the longest linkage group, which covers a genetic distance of 91.91 cM, ‘DMQ15’ has the shortest linkage group, which covers a genetic distance of 10.38 cM; the molecular linkage map of ‘Qiujinxing’ has 18 linkage groups with 89 markers, which covers a genetic distance of 529.58 cM, with an average interval of 5.95 cM. The average length of the linkage groups is 29.42 cM, The average number of loci per group is 4.94. Among the 18 linkage groups, ‘QJX1’ has the most loci (38) and the one with the fewest contains only 2. ‘QJX1’ also has the longest linkage group, which covers a genetic distance of 43.57 cM; the shortest is only 10.54 cM.

Figure 3. The genetic linkage map of ‘Damianqiu’.

Figure 4. The genetic linkage map of ‘Qiujinxing’.

According to a comparison of the two parents, many loci on the 1, 6, 8, 10,12, 15, 16, 18 linkage groups of ‘Qiujinxing’ correspond to the 1, 3, 4, 5, 8, 11, 13, 14, 18, 20 linkage groups of ‘Damianqiu’. QJX1 and DMQ1, DMQ3, DMQ4 have 37 shared markers (e2m21–400, e6m21–400, e5m22–195, etc.); DMQ8 and QJX15 have 2 shared markers (e22m20–1300, e22m20–680); DMQ11 and QJX6 have 4 shared markers (e17m17–250, e5m21–300, e13m17–270, e2m8–295); DMQ13 and QJX8 have 3 shared markers (e13m17–200, e13m17–440, e13m17–420); DMQ14 and QJX10 have 2 shared markers (e16m17–350, e16m17–255); QJX18 and DMQ18 have 2 shared markers (e15m17–170, e3m21–150); QJX20 and DMQ12 have 2 shared markers (e6m21–350, e6m21–215); QJX16 and DMQ5 have 2 shared markers (e14m16–370, e18m18–190). The data showed the high homology between these linkage groups. In future, we will add the co-dominance marker like SSR to make the integration of the two parents, and increase the density and uniformity of the map.

Conclusion

In this study, we used SRAP molecular markers to construct the first molecular genetic map of hawthorn in the world, as 18 linkage groups on male ‘Qiujinxing’ and 21 linkage groups on female ‘Damianqiu’ were obtained. Because the molecular markers were only few, the density of some linkage groups was not high. The chromosome number of hawthorn is 17, so the number of the linkage groups is 17 in theory. In the study, we obtained 18 linkage groups of male and 21 linkage groups of female. This indicates that at least in four chromosomes exist frequent commutation or gap. We are going to conduct more SRAP marker analysis, or increase other marker analysis such as SSR, SNP and ISSR. We can utilize complementarity between SRAP and SSR, ISSR, SNP to improve the density of the map and supplement the gap.

Many studies have reported the segregation distortion phenomenon in the construction of molecular genetic maps of other crops or fruit trees. They thought segregation distortion is ubiquitous in the construction of a map.[18–21] Segregation distortion will not influence the QTL detection.[22] The segregation distortion and the segregation marker have almost the same mapping efficiency. If the segregation distortion is removed, the gene of the relevant segment in the linkage map could lose the linkage relationship.[23] The segregation distortion marker is used to conduct linkage analysis, if the linkage groups contain the *. In this study, some segregation distortion appeared in all kinds of segregation loci. We used segregation distortion markers to conduct the construction of a genetic map and marked them with * in the map.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The work was supported by the National Natural Science Funds of China [grant number 31101515].