AFLP and SCAR Markers Associated with Peel Color in Eggplant (Solanum melongena)

Agricultural Sciences in China

December 2009

2009, 8(12): 1466-1474

AFLP and SCAR Markers Associated with Peel Color in Eggplant (Solanum melongena) LIAO Yi1, 2 *, SUN Bao-juan2 *, SUN Guang-wen1, LIU Hou-cheng1, LI Zhi-liang2, LI Zhen-xing2, WANG Guoping1 and CHEN Ri-yuan1 1 2

College of Horticulture, South China Agricultural University, Guangzhou 510642, P.R.China Vegetable Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, P.R.China

Abstract Peel color is an important breeding objective for eggplant. Dark purple and purplish red are the most common colors in commercial eggplant cultivars. A co-dominant amplified fragment length polymorphism (AFLP) marker which was associated with the peel color (each in coupling phase to dark purple and purplish red) was found in studying the genetic diversity in 58 eggplant accessions (contained cultivars and wild relatives). The maker bands were sequenced and converted to SCAR marker, this polymorphism in sequence was from an inserted/deleted (indels) mutation. And DNA from 136 eggplant materials (inbred lines, F1, and wild relatives) were amplified with the designed SCAR primers as template, high correlation between the SCAR marker and peel color (dark purple and purplish red) was found. Then, bulked line analysis (BLA) combined with AFLP was further used to identify polymorphic fragments, and another six AFLP markers were tested and verified to be associated with peel color, which demonstrated that BLA was an useful method for identifying molecular markers of interested traits. In conclusion, these markers will facilitate the MAS (maker-assisted selection) of eggplant breeding for peel color. Key words: eggplant, peel color, bulked line analysis, AFLP marker, SCAR marker

INTRODUCTION Eggplant (Solanum melongena L.) is not only a kind of tasty food, but also with underlying medical value (Konczak et al. 2004). Eggplant is one of the top ten vegetables in terms of oxygen radical absorbance capacity (Stommel and Whitaker 2003; Wu et al. 2004; Hanson et al. 2006) due to a novel source of anthocyanin, which is a major phenolic in eggplant and is the most important antioxidants with a variety of physiological functions such as anti-mutagenesis, anticancer and vision improvement (Todaro et al. 2009; Philpott et al. 2009; Azevedo et al. 2007). The anthocyanin concentration in eggplant fruit peel was higher

than those in other Solanaceae crops such as potato, tomato and pepper (Helmja et al. 2007; de PascualTeresa and Sanchez-Ballesta 2008). Anthocyanin is an important pigment for the coloration in plants. There were some correlations between the fruit color and phenlolic contents (Kitsuda et al. 2005; Hanson et al. 2006), and great variation (3-20 times) in contents and proportions of phenolics in eggplants (Stommel and Whitaker 2003; Prohens et al. 2007; Raigón et al. 2008). There even was no anthocyanin in green and white eggplant accessions (Daunay et al. 2004; Azuma et al. 2008). Peel color is a complex trait, which has been subjected by several classical genetic analyses as a qualitative trait (Tigchelaar et al. 1968; Liu and Phatok 1992).

This paper is translated from its Chinese version in Scientia Agricultura Sinica. LIAO Yi, MSc, E-mail: [email protected]; Correspondence CHEN Ri-yuan, Professor, Tel: +86-20-38294595, E-mail: [email protected] These authors contributed equally to this study.

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© 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60360-0

AFLP and SCAR Markers Associated with Peel Color in Eggplant (Solanum melongena)

Recently, some QTLs for color development in eggplant fruit have been mapped (Doganlar et al. 2002; Nunome et al. 2001). Fruit color in eggplant was quantitative trait in nature and was controlled by polygene, while the major gene heritability was dominant and the polygene heritability was subsidiary (Pang et al. 2008). The fruit color was affected by environmental factors (e.g., light and temperature) and cultivation conditions (Nothmann et al. 1978; Matsuzoe et al. 1999; Umeda et al. 2006). However, understanding of coloration genetics in eggplant is far less than in other Solanaceae crops (Jones et al. 2003; Paran et al. 2007; de Jong et al. 2004; Van der Knaap et al. 2006; Zygier et al. 2005; Ben Chaim et al. 2001). Bulked line analysis (BLA) was originally proposed by Tan et al. (1998), which was used for locating a Rf gene in rice, and verified by Zhao et al. (2004), who identified several SSR markers tightly linked to the Rf gene conferring fertility restoration of the cytoplasmic male sterility (CMC) Dian-type 1 in rice. Compared with bulked segregant analysis (BSA) (Michelmore et al. 1991), BLA has two main advantages for gene identifying. One is time-saving, instead of segregating progenies, conventional lines sharing the same phenotype can be bulked. Another is that the phenotypes of each line can be scored accurately, such as RILs by the replication of advance inbred lines under different growing conditions. Although with these advantages, contrast to BSA which reported in numerous papers, BLA still has rare report. So, the objectives of this study were: (1) to identify AFLP markers linked to the gene controlling peel color in eggplant; (2) to convert the obtained AFLP markers into SCAR markers to facilitate marker-assisted selection; (3) to demonstrate whether BLA can help to identify markers linked to the peel color in eggplant.

MATERIALS AND METHODS Plant materials 130 cultivated and 6 wild eggplant accessions, which were provided by Vegetable Institute of Guangdong Academy of Agriculture Science and College of Horticulture, South China Agricultural University, were

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used in this study. Most of the materials were advanced to inbred lines and breeding lines, while some resultant F1 progenies were also involved. These materials could represent the genetic diversity of eggplant in China. Experimental hybrids were included to obtain information on the heritability of the fruit color trait, which was of great relevant to the eggplant breeding program. The tested related species were S. torrum L., S. nigrum L., S. sisymbriifolium L., S. torvum Sw. (one accession each), and S. integrifolium P. (two accessions). Seeds were sown in a greenhouse, and then transplanted into field. Peel color was evaluated by eyes when fruit reached the commercial ripening stage.

DNA extraction and AFLP analysis DNA was extracted from leaves of 1-mon-old seedlings, ten samples each line were mixed with equal quantity as PCR template. The extraction procedure was conducted by CTAB method as described by Sun et al. (2008). AFLP procedure was performed essentially as described by Sun et al. (2008) with minor modifications. 100 ng DNA of each sample was completely digested with EcoR I and Mse I restriction enzymes, for 6 h at 37°C, and each adapter was ligated for 2 h at 20°C. The digested and ligated DNA was pre-amplified with the universal primers E0 and M0 (E 0: 5´-GACTGCG TACCAATTCA-3´; M0: 5´-GATGAGTCCTGAGTAAC3´). Selective amplification was performed with primers extended with two selective nucleotides (E0+2 and M0+2). Electrophoresis was performed in denaturing 6% (w/v) polyacrylamide gel (38 cm × 52.5 cm). After pre-running at 1 800V for 30 min, the samples were loaded and electrophoresed at 1 800 V for 2 h. Bands were detected by silver nitrate staining.

Cloning and sequencing of the target AFLP band The gel slice with polymorphic DNA fragment was cut with a razor blade and eluted with 20 —L ddH2O for overnight at 37°C. Then, 1 —L supernatant was used as template for re-amplification with the same primer combination and PCR condition as that of the selective amplification. The amplified products were sent to Shanghai Sangon Biological Engineering Technology and Ser-

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vice Co., Ltd., China, and BGI-Shenzhen, China, for sequencing, respectively.

Conversion of AFLP marker into SCAR maker and testing Based on the sequence of the cloned fragment, the EcoR I/Mse I restriction sites, two selective nucleotides, and the next internal sequences from both ends of the AFLP marker were used to design candidate SCAR primers. Two special primers each with 22 nucleotides were obtained and commercially synthesized, the name and sequence were as follows: SCAR-E: 5´-GAATTCAGCTACGATAAATAAG-3´ SCAR-M: 5´-TAACGGGTCATTACATGTTGGC-3´ The flanking markers were used to screen 136 different genotypes. PCR amplifications were performed in 20 —L reaction volumes containing 50 ng of genomic DNA of each genotype, 2 —L 10 × PCR reaction buffer, 2 mmol L-1 of MgCl2, 0.2 mmol L-1 of each dNTP, 20 ng of SCAR-E, 20 ng of SCAR-M, and 1 U of Taq polymerase (Shanghai Sangon Co., Ltd., China). The PCR reaction conditions were as follows: an initial denaturation step at 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 56°C for 60 s, 72°C for 30 s, and a final extension at 72°C for 30 s. The PCR products were electrophoresed through 6% (w/v) polyacrylamide gel as above mentioned.

Bulked line analysis Bulked line analysis was used to further identify candidate markers associated with the peel color in eggplant. Besides the color appearance, the previous AFLP and SCAR marker information were also as a criterion for construct procedure. Equal quantity of genomic DNA from 12 dark purple individual lines and 20 purplish red ones which present the bands that specific to the color was bulked, respectively, comprising of dark purple pool and purplish red pool. In addition, the bulked materials were chosen to cover a wide range of geographical origins so as to minimize other genetic background difference. The two pools were used as template for AFLP analysis. In terms of the former primer selection, 15 AFLP primer pairs (EcoR I/Mse I) were used to

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screen polymorphism between DNA bulks. Then, the candidate markers were verified by screening 69 accessions (Table 1).

RESULTS Peel color of eggplant accessions In the first eggplant accession group (Table 1), 20 accessions of fruit peel were black purple, 31 were reddish purple, 2 were deep reddish purple, 5 were white, and 11 were green; in the second group (Table 2), the accessions with black purple, reddish purple, white, green, bright red, and pink fruit peel were 27, 31, 3, 4, 1, and 1, respectively.

A co-dominant AFLP marker associated with peel color in eggplant 58 eggplant accessions (Table 1, 1-58) were used to analyze the genetic diversity, a co-dominant AFLP marker generated by primer pair E-GC/M-GG was significantly correlated with the peel color (Fig.1). The 2 fragments were named as E10M19-1 and E10M19-2. 13 of the 15 accessions with dark purple peel color presented E10M19-1; while only 4 of the 27 accessions with reddish purple presented the E10M19-1. The band E10M19-2 presented in the accessions was the inverse of E10M19-1.

Conversion of the AFLP marker into SCAR marker Sequence analysis results showed that the length of the fragment E10M19-1 and E10M19-2 were 108 and 107 bp, respectively (including the primer sequence). The 2 fragments were allele locus only with an inserted/deleted (indels) mutation the underline region (Fig.2). Based on the sequence information, two special primers were designed and used for PCR analysis of the DNA samples from individual lines (the former 58 accessions). PCR results revealed that most of the materials obtained the target band (single one), while 4 wild species had no amplified product, and fragment distribution was completely corresponded to the co-

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Table 1 List of the materials (1-69) used in the study and the distribution of the detected SCAR, AFLP markers among them Code

Accessions

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

05114 06005 06054-1 05110-2 05107-2-1 07006 06039-2 06017 V0159 96B-2 06014 07010 065810-1-2 07004CK 07011 065118-3-1 06061-1-2 06069-4 06071-2 06053-2 5119-3 06028 5553 5785 06112 V0150 V0099 V0467 V1010 V1172 05085-4 06073 05164 07008 06013 BoBai 2-2 06049-2-1 06015 5578 S. torrum 2) 05115 2) 05116 2) S. nigrum 2) V0139 V0200 V1463 0092 05098-2 YC212 05006 06113 06117 V0035 V1177 NanBeiLv 05155 5910 5811 V0130 08005-1

1) 2)

Fruit color Black purple Purplish red Purplish red Black purple Black purple Black purple Black purple Black purple Purplish red Purplish red Black purple Purplish red Purplish red Purplish red Purplish red Purplish red Purplish red Purplish red Purplish red Purplish red Black purple Black purple Deep purple-red Purplish red Purplish red Purplish red Purplish red Black purple Black purple Deep purple-red Purplish red Purplish red Purplish red Green Green White Purplish red Green Black purple Green Green Green Green Purplish red White Black purple Green Green White Purplish red Purplish red Black purple White Black purple Green Purplish red Green White Purplish red Purplish red

Markers 1) SCAR-1

SCAR-2

E 10M13

E 10M 18

E13 M10

E 13M 18

E4M9

E4M5

1 0 0 1 1 1 1 1 0 0 1 0 1 0 0 0 0 0 0 0 1 1 1 0 0 0 0 1 1 0 0 0 1 1 1 1 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0

0 1 1 0 0 0 0 0 1 1 0 1 0 1 1 1 1 1 1 1 0 0 0 1 1 1 1 0 0 1 1 1 0 0 0 0 0 1 0 0 0 0 0 1 0 1 1 1 1 1 1 0 1 1 1 1 0 1 1 1

1 0 0 1 1 1 1 1 0 0 1 0 1 0 0 0 0 0 0 0 1 1 1 0 0 0 0 1 1 0 0 0 1 1 1 1 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0

0 1 1 0 0 0 0 0 1 1 0 1 1 1 1 1 1 1 1 0 0 0 0 1 1 1 1 0 0 1 1 1 0 0 0 0 0 1 1 0 0 0 0 1 0 1 1 1 1 1 1 0 1 1 1 1 0 1 1 1

1 0 0 1 1 1 1 1 0 0 1 0 1 0 0 0 0 0 0 0 1 1 1 0 0 0 0 1 1 0 0 0 1 1 1 1 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0

0 1 1 0 0 0 0 0 1 1 0 1 0 1 1 1 1 1 1 1 0 0 0 1 1 1 1 0 0 1 1 1 0 0 0 0 0 1 1 0 0 0 0 0 0 1 1 1 1 1 1 0 1 1 1 1 0 1 1 1

0 1 1 0 0 0 0 0 1 1 0 1 0 1 1 1 1 1 1 1 0 0 0 1 1 1 1 0 0 1 1 1 0 0 0 1 1 0 0 0 0 0 0 0 1 1 1 1 1 1 0 0 1 1 1 0 1 1 1

1 0 0 1 1 1 1 1 0 0 1 0 1 0 0 0 0 0 0 0 1 1 1 0 0 0 0 1 1 0 0 0 1 1 1 1 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0

1 indicates the accession has the marker band; 0 indicates the accession has not the maker band; - indicates it’s not clear. The same as below. The accession is wild species. (Continued on next page)

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Table 1 (Continued from preceding page) Code

Accessions

61 62 63 64 65 66 67 68 69

27245-2-1 08008 07018-1 08005-2 2811 2805 05103 08005 05100

Fruit color Purplish red Black purple Purplish red Purplish red Black purple Black purple Black purple Purplish red Black purple

Markers SCAR-1 0 1 0 0 1 1 1 0 1

SCAR-2 1 0 1 1 0 0 0 1 0

E10M 13 0 1 0 0 1 1 1 0 1

E10 M18 1 0 1 1 0 0 0 1 0

E 13M10 0 1 0 0 1 1 1 0 1

E13 M18 1 0 1 1 0 0 0 1 0

E 4M9 1 0 1 1 0 0 0 1 0

E 4M5 0 1 0 0 1 1 1 0 1

Table 2 List of the materials (70-136) used in the study and the distribution of the co-dominant SCAR markers among them Code 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 1)

Accessions Chaozaolüqie Fasitekuaichangqie F1 S. sisymbriifolium L. 1) A69 Qibao, F1 A60 Helanbaiqie A68 Zihongchangqie 301, F1 Zaodaoshou 909 Hainanziqie Hainanxiaoyuanqie A74 27-2-0-2-123 Baiganlan Wandunjufengqie Xintiandihongchangqie, F1 Changyelang, F1 A45 A31 Sairuima Huimeichangqie Meizhoubao 303, F1 Texuanzichangqie A3 Jingqie 2, F1 A22 A51 Guilinmoqie Guangfeng 2, F1 A39 Meizichangqie, F1 Chenjindan Changqiuqie Moyuchangqie, F1 Diana, F1 Gongchang Lüguanqie Fenqie 3 S. torvum Sw. 1) Xiongyingchangbangqie, F1 Molongchangqie, F 1 Hunanxiaoyuanqie Ziguanqie Tianjinerminqie Beijingqiyeqie Youguanqiewang

The accession is wild species.

Fruit color Green Black purple Red Purplish red Purplish red Purplish red White Purplish red Purplish red Black purple Purplish red Purplish red Black purple Black purple Purplish red White Black purple Purplish red Black purple Purplish red Purplish red Black purple Black purple Purplish red Black purple Purplish red Black purple Purplish red Purplish red Black purple Purplish red Purplish red Mauve White Green Black purple Black purple Purplish red Green Purplish red Green Black purple Black purple Black purple Black purple Black purple Black purple Purplish red

SCAR markers SCAR-1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 1 1 0 1 0 1 0 0 0 0 0 0 1 0 1 1 0 1 1 1 1 1 1 1 1 1

SCAR-2 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 0 0 1 0 1 0 1 1 1 1 1 1 0 1 0 0 1 0 0 1 0 0 0 0 0 0 (Continued on next page)

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AFLP and SCAR Markers Associated with Peel Color in Eggplant (Solanum melongena)

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Table 2 (Continued from preceding page) Code

Accessions

Fruit color

118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136

Helanwujinqie Xinheizhenzhu Blackbounty A12 24-1-1-1-2 Blacknite A37 Taiguoxiaoqie Hunanxiaoqie × Blacknite A53 Hainanziqie × Blacknite A30 A21 Hainanziqie × Blackbounty A46 Zijiarenchangqie, F1 A32 Malaihong Malaihong × Blacknite

Black purple Black purple Black purple Purplish red Purplish red Black purple Purplish red Purplish red Black purple Purplish red Black purple Purplish red Purplish red Black purple Purplish red Purplish red Purplish red Purplish red Black purple

SCAR markers SCAR-1 1 1 1 0 0 1 0 0 1 0 1 0 0 1 0 0 0 0 1

SCAR-2 0 0 0 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 1

Fig. 1 The band pattern of the AFLP markers on some materials. M is DNA marker and the lane’s number are in accordance with the materials’ code from Table 1.

purple peel color, presented SCAR-2, only 2 presented SCAR-1. According to statistic analysis of 111 accessions with purple skin, the coincidence between the SCAR markers and peel color was more than 90%. Fig. 2 The sequence of E10M19-1. The shadow regions are primer sequences, the underline region is mutation locus.

dominant AFLP marker, which validated that the AFLP marker was successfully converted into SCAR marker. The two target bands were named as SCAR-1 and SCAR-2, respectively (Fig.3). DNA from other 76 accessions (59-136) were used to verify the association between the SCAR marker and peel color of eggplant. 24 of the 33 accessions with dark purple color presented SCAR-1, 4 presented SCAR-2, and 5 hybrids (F1) presented both; 32 of 34 accessions with reddish

Other AFLP markers associated with peel color identified by BLA Six polymorphic fragments were detected between the dark purple DNA pool and purplish red one by using 15 primer combinations. Of them, 3 in coupling phase to dark purple color, 3 to purplish red color. These makers were obtained from the primer pairs: E-AC/M-TC, E-GC/M-GC, E-AA/M-TG, E-AA/M-GC, E-AC/M-AC, and E-GC/M-CA, and named as: E4M9, E10M18, E13M10, E13M18, E4M5, and E10M13, respectively. Further PCR

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Fig. 3 The distribution of the SCAR markers among some materials. M, DNA markers, the lanes from 1 to 32 are in accordance with the materials’ code 1-32 in Table 1, and lane 33-48 are in accordance with code 121-136 in Table 2.

Fig. 4 AFLP polymorphisms obtained with some EcoR I/Mse I primer combinations in a bulked line analysis. The left line, purplish red pool; the right line, dark purple pool.

analysis of individual accessions (53 of 69 lines with dark purple or reddish purple peel color) showed that all of the 6 markers had high correlation to the peel color, and the coincidence rate was up to 85% (Table 1), this indicated that the 6 AFLP markers were correspondence to the SCAR marker, and associated to peel color in eggplant.

DISCUSSION Fruit color is one of the most variable morphological traits in eggplant, due to different pigments and distribution, fruit color can display diverse patterns (Daunay et al. 2004). While the color of most of comercial eggplant cultivars is various shades of purple (Sêkara et al. 2007). The SCAR marker was only associated with peel color that confined to purple background (reddish purple and dark purple), indicating that this marker might link to a modi-

fier gene which was hypostatic to be the basic genes (Tigchelaar et al. 1968) and quantitatively or qualitatively alter pigments concentration in fruit peel. The deep color of fruit was incomplete dominant to the light type in eggplant. This study was coincident to that not only by field observation but also analysis at molecular lever. The F1 plant from parents which both with dark purple or reddish purple peel only presented SCAR-1 or SCAR-2, respectively; while parents with dark purple and reddish purple, or with reddish purple and dark purple, the hybrids presented the 2 SCAR bands and their fruit color were dark purple, indicating that the SCAR marker was co-dominant in nature and correspond to the color performance indeed. In order to confirm whether this marker linked to a gene that determines the coloration in eggplant, a F2 population was being constructed. Linkage analysis would also be done based on this population. Sequence BLAST showed there was no high similarity sequence to it, so it was a new fragment in eggplant genomic reported to be associated with peel color. Whether the indels lead to a frame shift mutation in the coding region of a target gene is not known, and need further study. In our study, another 6 AFLP makers were identified and verified to associate with peel color in eggplant by BLA, demonstrating this method is useful for maker development in some specific condition. Several explanations may attribute to this succeed application in eggplant. First, S. melongena origin from the same progenitors, even after long time evolution and demonstration, the genetic diversity is still narrow, so the modern cultivars could be regarded as the recombinant inbred lines derivating from the same ancestor;

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AFLP and SCAR Markers Associated with Peel Color in Eggplant (Solanum melongena)

second, the lines selected for pooling strategy covered a rich range of morphological and geographic diversity and the number is enough to mask the interference of other traits; third, most of the materials were advanced inbred lines that had a robust and stable phenotype and the genetic background trending to be homozygous. In addition, the intensity of the purple pigments in fruit of eggplant may be controlled by few major genes or QTLs that display a high heritability. However, whether this method could widely apply to other plants is still unknown, and need more experimental verification.

Acknowledgements The work was jointly supported by the Research Fund of Higher Education of Guangdong Province, China (cgzhzd0406) and Agriculture Research Foundation of Guangdong Province, China (2007A020200005-2).

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