Introduction Because of late season, it may play a significant role in fruit market. However, its origin is still
1. Introduction Because of late season, it may play a significant role in fruit market. However, its origin is still
Citrus are one of the major fruit crops in the unconfirmed. SSR is a reliable method for parentage world [1]. Citrus varieties are related to the analysis on account of the co-dominant inheritance development of Citrus industry. In China, there are and large number of alleles per locus [3]. On the other many Citrus resources, and a large amount of genetic hand, cpSSR can clearly identify the female parentage variation exists within them. It is an important for the characteristic of female genetics [4]. The aim breeding way selecting the required Citrus varieties of this study was to clarify the possible parentage of from local resources [2]. In 1956, the fruit resources Huangguogan via the combination of morphological investigators discovered an obvious Citrus hybrid in and molecular markers with SSR and cpSSR. Shimian County, Sichuan Province, China and named
it as Huangguogan. Its fruit is egg round in shape,
2. Materials and Methods
surface colour is orange, has thick skin but easy to
2.1 Plant Materials
peel and seedless. Many morphological and physiological indicators range between the tangerine
In order to clarify the possible parentage of and the orange. Besides, it is suitable for Huangguogan, the Citrus varieties (Table 1) which transportation and export and ripens in March or April.
were cultivated longer than Huangguogan in Shimian County, Sichuan Province, China were collected.
Corresponding author: Zhi-Hui Wang, Prof., research fields: plant physiology, molecular biology technology. E-mail:
Shatian pummelo (Citrus grandis (L.) Osbeck) was [email protected].
Charaterization of Citrus Hybrid “Huangguogan” through the
Combination of Morphological and Molecular Markers
accepted to be control in the study.
2.4 SSR Analysis
2.2 Morphological Analysis Seventeen previously reported primer pairs were used (Table 2) [6, 7], synthesized by Sangon Inc.
Thirty spring leaves selected randomly were used to (Shanghai, China). The PCR reactions were performed
measure their leaf stalk length and phylliform index in a PTC-200 thermocycler (MJ Research, Waltham,
and thirty ripe fruit selected randomly were used to MA) in 20 µL reaction mixtures containing 10 uL
measure their fruit shape index. 2×Taq MasterMix (CW Inc., Beijing, China), 0.8 uM
2.3 DNA Extraction of each forward and reverse primer, 50 ng of template Total genomic DNA was isolated from fresh leaves
DNA and sterilized double stilled water. The following the procedure previously described by
amplification program consisted of an initial Cheng et al. [5]. The quality and concentration of the
denaturing cycle at 94 °C for 5 min; 30 cycles of 30 s DNA samples were determined by spectrophotometer,
(denaturing) at 94 °C; 45 s (annealing) at corresponding and the sample concentration was diluted to 50 ng·µL -1 .
temperatures (Table 2); 1 min (elongation) at 72 °C;
Table 1 The Citrus varieties provided for the study.
Accession Variety
Latin name
Collection site
1 Huangguogan Shimian County, Sichuan Province, China 2 Yaogan
Shimian County, Sichuan Province, China 3 Sweet orange
Shimian County, Sichuan Province, China 4 Hongju tangerine
C. sinensis (L.) Osbeck
Shimian County, Sichuan Province, China 5 Navel orange
C. reticulata Blanco
Shimian County, Sichuan Province, China 6 Shatian pummelo
C. sinensis (L.) Osbeck
Sichuan Agricultural University, China 7 Huangguogan older than 100 years
C. grandis (L.) Osbeck
Shimian County, Sichuan Province, China 8 Satsuma mandarin
Shimian County, Sichuan Province, China Yaogan is a wild Cirtus we discoveryed in Shimian County, Sichuan Province, China.
C. umshiu Marc.
Table 2 Simple sequence repeat (SSR) primer sequences provided for the study.
Annealing Accession Primer F-Primer (5 ′-3′) R-Primer (5 ′-3′) temperature (°C) Reference
1 TAA27 GGATGAAAAATGCTCAAAATG TAGTACCCACAGGGAAGAGAGC 55 Ref. [6] 2 ATC09 TTCCTTATGTAATTGCTCTTTG TGTGAGTGTTTGTGCGTGTG
55 Ref. [7]
3 TAA41 AGGTCTACATTGGCATTGTC ACATGCAGTGCTATAATGAATG 49 Ref. [6] 4 AAGG9 AATGCTGAAGATAATCCGCG TGCCTTGCTCTCCACTCC
49 Ref. [7] 5 CAC15 TAAATCTCCACTCTGCAAAAGC GATAGGAAGCGTCGTAGACCC 49
Ref. [6]
6 AG14 AAAGGGAAAGCCCTAATCTCA CTTCCTCTTGCGGAGTGTTC 49 Ref. [7] 7 TAA15 GAAAGGGTTACTTGACCAGGC CTTCCCAGCTGCACAAGC 49
Ref. [6]
8 CAC39 AGAAGCCATCTCTTCTGCTGC AATTCAGTCCCATTCCATTCC 55 Ref. [6] 9 TAA33 GGTACTGATAGTACTGCGGCG GCTAATCGCTACGTCTTCGC
55 Ref. [6] 10 CT19 CGCCAAGCTTACCACTCACTAC GCCACGATTTGTAGGGGATAG 55
Ref. [7] 11 GT03 GCCTTCTTGATTTACCGGAC TGCTCCGAACTTCATCATTG 51
Ref. [7] 12 CT02 ACGGTGCGTTTTGAGGTAAG TGACTGTTGGATTTGGGATG 51
Ref. [7]
13 CAG01 AACACTCGCACCAAATCCTC TAAATGGCAACCCCAGCTTTG 51 Ref. [7]
14 CAT01 GCTTTCGATCCCTCCACATA GATCCCTACAATCCTTGGTCC 51 Ref. [7] 15 TAA3 AGAGAAGAAACATTTGCGGAGC GAGATGGGACTTGGTTCATCACG 53
Ref. [6] 16 CT21 CGAACTCATTAAAAGCCGAAAC CAACAACCACCACTCTCACG 53
Ref. [7]
17 TC26 CTTCCTCTTGCGGAGTGTTC GAGGGAAAGCCCTAATCTCA 53 Ref. [7] F = Forward; R = Reverse.
Charaterization of Citrus Hybrid “Huangguogan” through the
Combination of Morphological and Molecular Markers
and one final cycle of 10 min at 72 °C. Samples were (1) and absence (0). Genetic relationships among the then stored at 4 °C. The amplification products were
haplotypes on the basis of SSR-amplified fragment separated by 12% polyacrylamide gel electrophoresis
sizes were analyzed with NTSYS-pc Version 2.10 (PAGE) and detected by silver staining method
software. A similarity matrix was generated by previously described by Federici et al. [8].
calculating the proportion of bands shared by each pair of accessions (Jaccard coefficient), and a
2.5 cpSSR Analysis dendrogram was constructed using the unweighted
Four previously reported primer pairs were used pair group method based on arithmetic means (Table 3) [9], synthesized by Sangon Inc. (Shanghai,
(UPGMA) in Cluster Analysis.
China). The PCR reactions were performed in a PTC-200 thermocycler (MJ Research, Waltham, MA)
3 Results
in 20- μL reaction mixtures which were the same with
3.1 Morphological Identification
SSR analysis. The amplification program consisted of an initial denaturing cycle at 94 °C for 3 min; 32
Leaf stalk length, fruit shape index and phylliform cycles of 1 min (denaturing) at 94 °C; 40 s
index of Huangguogan were similar to Sweet orange (annealing) at 55 °C; 1 min (elongation) at 72 °C;
but were statistically different from the rest of the and one final cycle of 5 min at 72 °C. Samples were
varieties as presented in Table 4. Previous studies on then stored at 4 °C. The next steps were the same
anther and pollen morphology reported that anthers of with SSR analysis.
Huangguogan were plump and yellow and pollen of Huangguogan was plenty, similar to Sweet orange but
2.6 Data Analysis different from Navel orange. The pollen stain ability
The data of morphological analysis were calculated was also close to Sweet orange [10]. All together, using the software package SPSS V13.0. All clearly
these findings suggest that Huangguogan has detectable SSR products were scored as band presence
morphological similarities to Sweet orange.
Table 3 Chloroplast simple sequence repeat (cpSSR) primer sequences used in the study.
Accession Primer F-Primer (5 ′-3′) R-Primer (5 ′-3′) Reference
1 SPCC1 CTTCCAAGCTAACGATGC CTGTCCTATCCATTAGACAATG Ref. [9] 2 SPCC3 GATGTAGCCAAGTGGATCA TAATTTGATTCTTCGTCGC
Ref. [9]
3 SPCC9 TAAAGAAGGTTCTTTTTCAAGC CGAACCCTCGGTACGATTAA Ref. [9] 4 SPCC11 GGCCATAGGCTGGAAAGTCT GTTTATGCATGGCGAAAAGG
Ref. [9] F = Forward; R = Reverse.
Table 4 Analysis of leaf stalk and phylliform index and fruit shape index.
Accession Variety
Leaf stalk length/cm
Fruit shape index
Phylliform index
1 Huangguogan
1.84 ± 0.14 a 1.024 ± 0.006 b 2.12 ± 0.04 a
2 Yaogan
0.43 ± 0.08 d 0.768 ± 0.004 c 2.03 ± 0.05 b
3 Sweet orange
1.88 ± 0.12 a 1.042 ± 0.003 b 2.15 ± 0.06 a
4 Hongju tangerine
1.49 ± 0.14 b 0.794 ± 0.004 c 2.02 ± 0.05 b
5 Navel orange
1.42 ± 0.09 b 1.114 ± 0.008 a 1.91 ± 0.07 c
6 Shatian pummelo
1.22 ± 0.10 c 1.118 ± 0.007 a 1.86 ± 0.09 c
7 Huangguogan older than 100 years
1.78 ± 0.13 a 1.032 ± 0.005 b 2.10 ± 0.04 a
8 Satsuma mandarin
0.82 ± 0.08 c 0.786 ± 0.005 c 2.01 ± 0.05 b
Same letter in a column means no significant difference at P ≤ 0.05. Number refers to the samples listed in Table 1.
Charaterization of Citrus Hybrid “Huangguogan” through the
Combination of Morphological and Molecular Markers
3.2 SSR Characterization
3.3 cpSSR Identification
Twelve out of seventeen SSR primer pairs were Two out of four cpSSR primers were polymorphic polymorphic among these varieties. Gel image picture
(Figs. 3a and 3b). Band patterns were identical in showing the amplicons of seven varieties amplified by
Huangguoggan (Lane 1), Sweet orange (Lane 3), SSR primer TAA1 is shown in Fig. 1. It clearly
Navel orange (Lane 5) and Shatian pummelo (Lane 6); demonstrated that allelic was similar between however, they were different from Yaogan (Lane 2), Huangguoan (Lane 1) and Hongju tangerine (Lane 4)
Hongju tangerine (Lane 4) and Satsuma mandarin and allelic variation among others. These results
(Lane 8) in Figs. 3a and 3b. Since cpSSR is maternal reflected obviously that the SSR was a useful tool in
inheritance, together with the morphological data it is distinguishing these genotypes.
likely that the female parentage of Huangguoggan Consequently, SSR results were collectively used to
could be Sweet orange.
construct UPGMA dendrogram to reveal genetic When the findings of the present study were relationships among varieties. In the cluster analysis,
taken into consideration, SSR data and the dendrogram the dendrogram had two main branches, first branch
including all of the varieties except Shatian pummelo which formed a separate branch. Huangguogan was clustered together with Hongju tangerine and revealed ~80% genetic similarity (Fig. 2). Huangguogan is the closest to Hongju tangerine, and Sweet orange and Navel orange are together and ~60% similar to
(a) Huangguogan.
(b)
Fig. 1 SSR profile of eight Citrus accessions using primer Fig. 3 cpSSR profile of eight Citrus varieties using primer pair TAA1. Number refers to the samples listed in Table 1;
pair SPCC1 (A) and SPCC3 (B). Number refers to the M is size marker.
samples listed in Table 1; M is size marker.
Fig. 2 Dendrogram (UPGMA) representing genetic relationships among eight Citrus based on the dice coefficient obtained from pooled allelic profile of SSR markers technique; number refers to the samples listed in Table 1.
Charaterization of Citrus Hybrid “Huangguogan” through the
Combination of Morphological and Molecular Markers
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4. Discussion and Conclusions
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Apr. 2013, Vol. 7, No. 4, pp. 353-357 Journal of Life Sciences, ISSN 1934-7391, USA
A Test for Stabilization of an Oligomeric Protein by Introduction of Aromatic Residues into the Interface
Yuho Mano, Ayako Shiota, Kotaro Hara, Azumi Hirata, Masayuki Oda and Kazufumi Takano Department of Biomolecular Chemistry, Kyoto Prefectural University, Kyoto 606-8522, Japan
Received: December 14, 2012 / Accepted: February 5, 2013 / Published: April 30, 2013.
Abstract: The design of variants to enhance conformational stability of proteins is an important aspect of protein engineering. Oligomeric proteins are often stabilized by aromatic clusters located within the subunit interfaces. In the present study, the authors constructed five variants of Ps3 αHSD (Pseudomonas sp. B-0831 3-hydroxysteroid dehydrogenase) in which one or two residues at the dimer interface were replaced with aromatic residues, and examined the effects of introducing aromatic residues in this region on protein thermostability. Under their experimental conditions, all variants formed dimers, similar to wild-type Ps3 αHSD. Thermal denaturation experiments indicated that T m of all variants was 0.2-16.2 ºC lower than that of wild-type protein, indicating less stable thanwild-type protein. The results collectively suggest that aromatic residues of natural oligomeric proteins are strictly posted in the interface to facilitate optimal interactions and avoid conformational strain.
Key words: Pseudomonas sp. B-0831 3 -hydroxysteroid dehydrogenase, oligomeric protein, conformational stability, aromatic residue.