Molecular Plant Breeding 2016, Vol.7, No.27, 1
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individuals to carry out multiple sequence alignment and clustering analysis, and then merges an individual into a
certain taxonomical group accurately. The following short homologous DNA sequences were commonly applied
in the plant barcoding: code region fragment of chloroplast genome such as
rpoB
(subunit of RNA polymerase b),
rpoC1
(ribonucleic acid polymerase C1 subunit),
matK
(maturase K protein),
rbcL
(1,5 diphosphate
ribulose-1,5-bisphosphate carboxylase) and non-coding region fragment such as
trnH psbA
(plastid trnH psbA
spacer), fragment of the nuclear genome ITS (ribosomal internal transcribed spacer region) (Yan and Yu, 2010).
TrnH-psbA
sequence is a fragment of the chloroplast space (non-coding region), and its two sides contain a
conservative sequence of about 75 bp, which can be used to design universal primers, yet this sequence has a high
frequency of insertion and deletion; besides, the length differences of amplification among different species are
significant, so that this homologous DNA sequences has a good recognition for the allied species (Lahaye et al.,
2008). But so far we are unable to find any report about the application of DNA barcoding technology based on
trnH-psbA
sequence to analyze the phylogenetic relationship among lily systems. In order to provide a theoretical
reference for the combinations of lily crossbreeding, we used
trnH-psbA
barcode technology to study the
phylogenetic relationship among 67 lily germplasms, as well as to determine its fitness on interspecies and
intraspecies classification in this research.
1 Results and Analysis
1.1 Electrophorogram of lilies amplified based on
trnH-psbA
gene sequence
Amplified products were detected by 1% agarose gel electrophoresis. The results showed that this primer could
reach 100% amplification efficiency (Figure 1) and generate clear and stable fragments with the sizes ranged from
400 bp to 500 bp in length.
Figure 1 Agaros electrophorogram of lily in part based on trnH-psbA gene sequence
Note: M: DNA ladder marker 1500
1.2 Sequence alignment of lily based on trnH-psbA gene sequence
Homologous alignment of the sequencing results of the trnH-psbA barcode in 67 samples were carried out by
DANMAN. The results indicated that there be certainly differences in the length of trnH-psbA sequence in lilies.
Total sequence length is 460 bp after sequence alignment, which implied that there be happens of insertion,
substitution and deletion obviously. There were 415 conserved sites and 45 variable sites detected in this research.
1.3 Construction of phylogenetic tree of lilies based on chloroplast
trnH-psbA
gene sequence
The phylogenetic tree of 67 lily samples was constructed by PHYLIP. Figure 2 showed that Yebaihe NO.28
(
Lilium brownii)
(germplasm origin from Lichuan Baiyang Town) and Yebaihe NO.62 (‘White Heaven’×
Lilium
bakerianum
var.
delavayi
)) first formed a new branch and were classified into a group respectively from the
bottom of the tree. Then, Baihua Baihe NO.30 (
Lilium brownii
var.
viridulum
) was also divided into a group and
formed a single branch, and Chuanbaihe NO.25 (
Lilium davidii
) (germplasm origin from Yulong Snow Mountain)
was separated as well, indicating that the above 4 lily samples had farther relationship with the rest 63 samples.
And then, we classified the rest 63 tested samples into five groups. The 24 samples on the top were classified into
the first category (Category
Ⅰ
), which mainly consist of cultivated sample, and Category
Ⅰ
can be further divided
into 2 subgroup (Ia and Ib). Subgroup Ia contained 10 lily samples, including Chuanbaihe No.33 (L. davidii )
(germplasm origin from Xizhou Town, Dali City) and Chuangbaihe No.34 (L. davidii) (germplasm origin from
Yulong xueshan, Lijiang City), both of them were the primitive parents of Asian lily (Lilium asiatic). Hybrid
offspring No 65 of Pollyanna’×Lilium dauricum came from the cross of Asian lily (L. asiatic ) and Maobaohe (L.
