Application of RAPD molecular markers for groups of inbred lines by
[Papers] ZHAO Jiu-Ran, Guo Jinglun, Guo Qiang, Wei Deming, Kongyan Fang, Zhao Jiuran, Guo Jinglun, Guo Qiang, Yu Deming, Kong Yanfang - Agricultural Technology understand.
The following is a draft at the time, cluster map is hand painted, and there is no formal post there!
application of RAPD molecular markers for key groups of inbred lines of maize
Research by
Abstract: RAPD markers for 25 of the current genetic relationships of maize inbred lines were divided into groups. This study established from seed, bud and leaf tissue DNA extraction method of trace . RAPD primers from box A to O test a total of 300 primers were screened with a PCR product of maize polymorphism primer 40, which was particularly evident with the 10 polymorphic primers. They are F3, O20, A19 , M2, M6, N11, N12, N19, C7 and G14 and so on. 10 primers according to established bands of 0,1-type data to calculate the genetic distance between the 25 inbred lines, and cluster analysis. The results show that: tested a total of 25 inbred lines can be divided into 5 groups. I class Sipingtou blood system: including the Huang early four, Kyrgyzstan 853, yellow wild four, four from four, 196,81515,404, H21, etc. 8 a; II Reid Yellow Dent class blood system: including 478,488,3189,7922,8112, B sharp 8,5005 a total of seven inbred lines, etc.; Ⅲ class system for the blood of Lancaster include: MO17, As early as 49, 22 so a total of more than 3; Ⅳ class Ludahonggu blood system: only one inbred line E28. The first class Ⅴ total of P78, 9502,178, P138, 007, P17 and other six inbred lines. This group are hybrids from the United States 78599 Progeny from. and two other groups the United States Reid Yellow Dent and Lancaster system between the system far. crosses between them most of the optional hybrid with a strong advantage. This group of inbred lines of maize breeding and production in China on the application will be further expanded. The results showed that: Maize RAPD molecular markers and pedigree wears Grouping agreement. by hybridization between different inbred lines , the yield performance of hybrid vigor, but also verify the application of RAPD markers by five different groups of the correctness of kinship.
Key words: RAPD groups of maize inbred lines divided by
accurate affinity groups, to establish taking advantage of the corresponding hybrid model, targeted matching crosses, will greatly reduce the breeding of blindness and improve breeding efficiency. in the past often used by foreign scholars group methods are: (1) Difference between morphological characteristics according to the method vulnerable to environmental conditions; (2) based on geographical origin and division of pedigrees. But geographical distance does not necessarily exist between inbred heterosis, of unknown origin or source of the pedigree of germplasm difficult to accurately divide the complex; (3) through a large number observed with the parental ability to pay the performance test to determine that this method takes a lot of manpower, material resources, and time consuming; (4) isozyme technology to provide a way to divide groups. But it provides genetic loci is limited, difficult to distinguish between many materials; (5) RFLP markers. to provide a more extensive polymorphism, to overcome the appeal of the defect, has been used successfully by groups of maize inbred lines (5), (6). The results consistent with the pedigree. However, because of complicated operation, technical complexity, high cost, typically using isotope labeling, are not easily mastered by the majority of corn breeders, and come from behind because of RAPD markers is simple, small amount of sample, polymorphism of the rich, environmental conditions and developmental stages from the impact of the economic advantages of fast and is widely used. Fukuoka (13), who used 28 primers of 16 rice varieties were RAPD amplification reactions and put them into three categories , and consistent with the conclusions of RFLP classification. Yang and Quiros (11), who use the technology classification of celery satisfactory results are obtained in the corn, Xin-Zhi Liu (2) and others with 6 to 15 RAPD primers maize inbred lines were divided into groups, the results consistent with the pedigree method. In this study, RAPD molecular markers of inbred lines and part of our excellent choice system, a total of 25 study materials were divided into groups, from the molecular level for the use of corn breeders group with good heterosis hybrid model provides a basis.
1, Materials and methods
1-1
tested materials used in this study of 25 maize inbred Department of maize production in our country a representative part of the backbone system and breeding in recent years, the advantages of the system unit. All materials were tested after 2 years of field observations, proved homozygous genetically stable, consistent and tidy, and bagging since pay. examination Inbred name, number and pedigree experiments in Table 1
1-2
1-2-1 DNA extraction using Guojing Lun, etc. (3) improved seeds of maize single-DNA extraction New Methods: dry seeds, will take off the seed embryo, into the 1.5ml centrifuge tube, add 100ul chloroform grinding, then add 300ulDNA extract (100mM Tris-Hcl, 50mM EDTA, 50mM Nacl, 1.5% SDS ) were mixed after the 10,000 rev / separation heart 2
minutes, the supernatant by adding pre-absorption of anhydrous ethanol with 500ul 1.5ml centrifuge tubes,
Table 1
number
inbred Name
pedigree
1
478
8112t5003
2
488
5003t8112
3
3189
5003t8112
4
5005
American hybrids as early as 3147
5
yellow
Tangsipingtou four natural variants
6
yellow wild four
(early yellow pheasant red t d) t baidunzi Huang
7
four from four as early as four yellow t
from 330
8
404
early as four yellow t Mexico Department of
9
853
early as four yellow t from 330
10
H21
Huangzao four tH84
11
196
Huangzao four t340
12
81515
(100t short C103S2 Fung Wah ) t Huangzao four
13
E28
tour the United States Department of A419HTt 9 W 14
7922
U.S. Hybrids 3382
15
8112
U8 U.S. corn hybrids
16
than the tip of eight
(B73t tip 2) t8112
17
P78
U.S. corn hybrids P78599
18
007
U.S. corn hybrid species P78599
19
P17
U.S. corn hybrids P78599
20
9501
U.S. corn hybrids P78599
21
early 49
22
more than 22
23
178
U.S. corn hybrids P78599
24
P138
U.S. corn hybrids P78599
25
Mo17
187-2tC103
back and forth gently upside down 2 times, 12,000 rev / 1 min isolated hearts, drained supernatant, add 70% ethanol washed, air dried directly into 100ul TE, to be fully dissolved supernatant spare.
1 -2-2 RAPD reaction conditions and detection methods
RAPD amplification products in the PTC-100PCR reaction is carried out instrument, the OPERON primers produced for the United States, length of 10bp. The reaction program was: 940C denaturation for 1 minute, 940C denaturation for 20 seconds, 370C combination of primers and templates for 1 minute, 720C heat for 5 minutes, to extend more fully.
reaction system: Each 25ul reaction solution including the 10m mol / L Tris-Hcl (pH8.0), 50m mol / L Kcl, 2m mol/LMgcl2, gelatin 0.001%, 4tdnTps were 0.2m mol / L; primer 1umol / L, 1.5 units TaqDNA polymerase, 40ngDNA template.
PCR products were 1TAE buffer conditions, 1.2% agarose gel electrophoresis, ethidium bromide staining, observed under ultraviolet light photography.
1-2-3
statistical methods can be distinguished for each RAPD amplified products represent a site of amplified bands per locus based on the availability of the statistics, there are counted as 1, no count is 0. sample distance formula:
dij = b / (a + b) t100
a: that two species have the same number of bands
b: two species that have not the same distance between bands of
class are classified by group average method to calculate the computer to complete.
2, the results Analysis
2-1 RAPD markers determination results
from 300 primers amplified a product of the 25 inbred lines with 40 polymorphic primers, accounting for 13.3% of primer. with a particularly marked 10 polymorphic primers, they are: F3, O20, A19, M2, M6, N11, N12, N19, C7 and G14 and so on. The primer sequences in Table 2. on the 25 inbred lines in the same location on the DNA fragments , a RAPD band recorded as 1, Wuji 0. See Figure 1 and Figure 2 example. the 10 primers with 0-1 type results into the data.
Table 2 of 25 maize inbred Department of polymorphism is particularly obvious
10 primers
primer sequences (5, -3,)
primer sequences (5, -3,)
A19 < br> CAAACGTCGG
M06
CTGGGCAACT
C07
GTCCCGACGA
N11
TCGCCGCAAA
G14
GGATGAGACC
N12
CACAGACACC
F03
CCTGATCACC
N19
GTCCGTACTG
M02
ACAACGCCTC
O20
ACACACGCTG
2-2 25 maize inbred lines to the above cluster analysis of RAPD analysis of the results obtained
0 , 1 data, using cluster analysis statistical software calculated the genetic distance among 25 inbred lines included in Table 3.
can be seen from Table 3, 25 inbred lines between the genetic distance between 1.59 ~ 36.51 . genetic distance between 478 and 488, both from the same second ring system. inbred genetic distance is greatest between 178 and 7922. Although both are hybrids from the United States, but are P78599 and 3382 offspring. for different groups.
inbred lines according to the distance between clustering by group average method, the results shown in Figure 3 .25 copolymer inbred lines into five categories. Class 1 includes four yellow early, Kyrgyzstan 853 , yellow wild four, four from four, 196,81515,404, H21, etc. 8 inbred lines; Category 2 includes 478,488,3189,7922,8112 than eight-point, 5005, totaling seven inbred lines; Class 3 includes the Mo17, as early as 49, 22 so a total of more than 3; E28 Class 4 only one inbred line; Category 5 Total P78, 9502,178, P138, 007, P17 and other six inbred lines.
Table 4
Red
Ludahonggu
Lancaster
Tangsipingtou
Ludahonggu
21.0886
Lancaster
25.3944
25.9267
Tangsipingtou
21.7961
27.2050
27.0500
P78599 generations
25.9636
28.8250
26.0139
26.6535
New Note: This table data is then hand counted overtime at night.
3, to discuss
threshold is 4.5922 divided groups, then T = 21, 25 inbred lines can be divided into five groups. This division results pedigree is consistent.
as in the first category I, Kyrgyzstan 853, yellow wild four, four from four, 196,81515,404, H21, etc are made as early as four pro-containing yellow blood of the second ring of the Department of Breeding from. so naturally, as early as four kinship with similar yellow. the combinations between these systems will not have a strong combination of excellent produce. drawn in group II, the 478,488,3189,7922,8112 than the tip August, 5005, etc., check all of its pedigree and an American Red blood BSSS group related to a typical Red system. Section III group of Mo17, as early as 49, more than 22 were among 21 genetic distance within the greater than the distance between the other groups is a typical Lancaster 25.Mo17 blood system, so these three lines are the Lancaster system. E28 inbred lines from the other groups were 25 or more, self-contained system , is a typical Ludahonggu system. It is important to point out that the first V class, and they are from the U.S. corn hybrids P78599 Progeny, four of them with the blood of the original system Tangsipingtou system, Red system, Lancaster Department of Ludahonggu Department of the genetic distance were 26 or more, we call P group. This group of inbred lines with other types of groups are likely to generate strong superior hybrid combinations. This is the introduction of China in recent years new germplasm and maize breeding and production in China play an increasing role. as in recent years, a hybrid of several outstanding Nongda 108 (P178t yellow C), CAU 3138 (Comprehensive 31tP138) and so on. Corn breeders for the future, the use of P group and the other four groups of priority system will be optional combination of superior maize hybrids strong main mode.
results of this study demonstrate: the use of RAPD molecular markers and has been the result of Grouping consistent with known pedigree back, RAPD markers in the group division of maize inbred lines is feasible. use this method in a wider range of maize inbred lines in China by research groups.
No comments:
Post a Comment