Genetic Diversity of Source Germplasm of Upland Cotton in China as Determined by SSR Marker Analysis
CHEN Guang, DU Xiong-Ming①
Key Laboratory of Cotton Genetic Improvement of Agricultural Ministry, Cotton Research Institute, Chinese Academy of Agricul-tural Sciences, Anyang 455004,China
Abstract: The genetic diversity of 43 sources of Upland cotton germplasm with different parental origins, breeding periods, and ecological growing areas in China were studied on the basis of simple sequence repeat (SSR) markers. A total of 130 gene alleles with 80% polymorphism were detected from 36 SSR primers. The number of alleles per primer ranged from two to eight with an average of 3.6. The polymorphism information content (PIC) range was 0.278-0.865, with an average of 0.62. The average geno-type diversity index (H’) was 1.102, the highest was 2.039 and the lowest was 0.451. The average coefficient of the genetic similar-ity of SSR markers among source germplasm was 0.610, ranging from 0.409 to 0.865. These indicated that the genetic diversity at the genomic level of the selected source germplasm was rich, and was representative of the diversity of the germplasms, in general. The diversity at the genome level of the base germplasm from the second and third breeding periods was decreased compared to that of the first period, indicating that the cotton genetic background in China became narrow gradually. The diversity of SSR markers among the base germplasm from early maturity cotton growing areas in the north was higher than those from the Huanghe and Yangtze growing areas. The molecular marker genetic similarity index of the domestic varieties was higher than that in the introduced varieties, which indicates that the genetic diversity in domestic cultivars was lower than that in the introduced varieties. This study gives an overview of the genetic diversity of the cotton germplasm base in China, and provides a guide for breeders to develop new cultivars efficiently.
Key words: source germplasm; SSR; genetic diversity
Source germplasm for cotton (Gossypium hirsu-tum L.) breeding in China includes germplasms with stable genetic characters, excellent yield, adaptability, and better general combining ability, and have been used frequently as parents in many breeding pro-grams. There are few reports on source germplasm research. Huang [1] described in the book “Cotton Variety and Pedigree in China” 36 source germplasms from the G. hirsutum varieties, comprising 25 foreign varieties, eight Chinese varieties and three varieties with low gossypol. Du and Liu [2] described a new method of classification of source germplasm by emphasizing the number of derived varieties in addi-tion to their pedigrees. The authors defined the source Received: 2005-09-07; Accepted: 2005-12-19
germplasm as being lines or varieties from which 20 or more applied varieties have been derived. Accord-ing to this criterion, 47 germplasm sources of Upland cotton used in China were described, which include 16 foreign varieties, 29 Chinese varieties, and two varieties with low gossypol.
Genetic diversity is the basic portion of biological diversity and is the base of biological polymorphism and species diversity. Genetic diversity and parenthood of germplasm play an important role in cotton breeding. The precise evaluation of the genetic diversity of excellent germplasm will provide a guide for choosing parents and predicting the degree of inheritance, variation, and level of heterosis, which are essential for realizing the breeding
This work was supported by the 10th Five Years Key Programs for Science and Technology Development of China (No. 2004BA525B05).
① Corresponding author. E-mail: ducri@public.ayptt.ha.cn; Tel: +86-372-2525 352
734 遗传学报 Acta Genetica Sinica Vol.33 No.8 2006
goal. Molecular marker analysis is a modern technique,
which discloses genetic differences at the DNA level in plants and is an effective tool for testing genetic diversity of germplasm in breeding programs [3, 4]. The study of cotton germplasm diversity has expanded from the phe-notypic, cellular, and biochemical levels to the DNA level. Modern molecular marker techniques can illuminate the individual differences and relationships among species at the DNA level. Most cotton varieties planted in China were derived from a few sources of germplasm such as DPL, Stoneville, King, Uganda, Foster, and Trice, all of which were introduced from abroad. These varieties were the foundation of Chinese cotton breeding programs and played a decisive role in the self-breeding varieties of China. It has been indicated that the genetic base was narrow and the genetic diversity was low in Upland cot-ton in China. This was caused by a limited quantity of source germplasms. We will present evidence from mo-lecular marker analysisshowing the correlation among source germplasms’ genetic diversity. The method using simple sequence repeat (SSR) markers is a fast and con-venient molecular marker method with good reproduci-bility and high veracity, which can be used to evaluate cotton varieties. Genetic similarity and clustering analyses of source germplasm at the molecular level were carried out in this study to provide some data for parent selection, germplasm enhancement, and application in our cot-ton-breeding program. The collection, conservation, and utilization of large collections of crop germplasms have aroused people’s interest for a long time. To solve the problem of the difficulty for finding source germplasm, research of core collections of wheat (Triticum aestivum L.), rice (Oryza sativa L.), and soybean (Glycine max L.) crop germplasms was done, and it is necessary to construct a core collection for cotton in China as there are more than 7 000 acces-sions in the Chinese cotton collection. The study on source Upland cotton germplasm will provide a base for identifying a core collection of Chinese cotton.
were used in this experiment (Table 1). They were grouped using three methods. One method divided the germplasm according to their three breeding periods — the first period (before 1950s), the second period (1950s-1960s), and the third period (1970s-1980s). The second method grouped germplasm on the basis of three Chinese growing areas—Yangtze river valley area, Huanghe river valley area, and the area in north-ern China. The final method divided germplasm into two groups on the basis of their origins — those bred in China and those introduced from abroad. 1. 2 SSR molecular marker analysis
Cotton genomic DNA was extracted on the basis of Zhang’s [5] CTAB method with some modifications [6,7]. Three hundreds and ninety-eight SSR primers, which were chosen on the basis of previous studies and po-tential polymorphism, were used to make a primary survey among eight varieties. From this survey, 78 polymorphic primers were identified. Thirty-six primers with good amplification, clear gel bands, strong signal, and clear background were used in the PCR reactions. A 10 μL volume of PCR reaction mixture contained 1.0 μL 10 × PCR buffer (contain-ing 20 mmol/L Mg2+), 0.2 μL dNTP (10 mmol/L), 0.3 μL Taq enzyme (2 U/μL), 6.2 μL ddH2O, 0.65 μL forward primer (5 μmol/L), 0.65 μL reverse primer (5 μmol/L), and 1 μL DNA template (50 ng/μL). The PCR reaction was carried out with a PTC-100 ther-mocycler (MJ Research, Waltham, USA). Amplifica-tion was programmed for pre-denaturing at 94℃ for 3 min, 30 cycles of denaturng at 94℃ for 30 s, an-nealing at 57℃ for 30 s, and extension at 72℃ for 45 s, followed by a final extension at 72℃ for 7 min. PCR amplification products were separated by elec-trophoresis using 8% nondenaturing polyacrylamide gels and were visualized following silver staining [8]. 1. 3 Data analysis
After observing the PCR electrophoresis results, the bands of DNA fragments were scored as present (1) or absent (0). Genetic diversity analyses were made on the basis of these scores. The statistical methods
1 Materials and Methods
1. 1 Plant materials
Forty-three Upland cotton source germplasms
CHEN Guang et al.: Genetic Diversity of Source Germplasm of Upland Cotton in China as Determined by SSR Marker Analysis 735
Table 1 Source germplasm and relative information No. Name Ecological area1 DPL14A 2 DPL15 3 4 5
Stoneville 4 Stoneville 4B Stoneville 2B
Origin
Parent name
Breeding period[2]
1 1 1 1 1
1 1 1 1 1
1 2 2 2
2 2 2
2 2 2 2 2 2 2 2 2
2 2
2 2 2 2 2 2 2 2
3 3 3 3 3 3 3
America America America America America America America
America America America
DPL14A DPL15 Stoneville 4B Stoneville 2B
Coker100 Delfos531 Empire Foster 6 King
Trice DPL14A DPL14A DPL14A
DPL15A DPL15A DPL15A
DPL15A DPL15A DPL15A DPL15A DPL15A Stoneville 4 Stoneville 4 Stoneville 2B Stoneville 2B Uganda Uganda
Delfos531 Foster 6 Foster 6 Stoneville 14A
6 Coker100 7 Delfos531 8 Empire 9 10 12 13 14 16 17 19 20 21 22 23 24 25 26 28 30 31 32 33 34 35 36 38 39 40 41 42 43
Foster 6 Guan nong 1 Jiangsu mian 1 Jiangsu mian 2 Jiangsu mian 3 Dong ting 1 Guang ye dai zi mian Zhong mian suo 2 Zhong mian suo 4 Zhong mian suo 3 Shan mian 4 Shan mian 5 Liao mian 3 Xu zhou 209 Xu zhou 1818 Zhong mian suo 7 Gan mian 1 Shan mian 3 Chao yang mian 1
Jin mian 2 Jin mian 1 Ke ke 1543 Yi shu hong Ji mian 1 Zhong mian suo 12 Hei shan mian 1 Zhong mian suo 10 Lambright GL-5 Micnarie 210
America America America America America America America North China Yangtze Yangtze Yangtze Yangtze America Huanghe Huanghe Huanghe Huanghe Huanghe North China Yangtze Yangtze Huanghe Yangtze Huanghe North China North China North China Russia Yangtze Huanghe Huanghe North China Huanghe America America
America Liaoning Jiangsu Jiangsu Jiangsu Hunan America Henan Henan Henan Shanxi Shanxi Liaoning Jiangsu Jiangsu Henan Jiangxi Shanxi Liaoning Liaoning Liaoning Russia Hubei Hebei Henan Liaoning Henan America America
11 Trice America America
15 57-681 Yangtze Sichuan
18 DPL16 America America
27 Uganda 4 29 52-128
Uganda Uganda Yangtze Sichuan
Chao Yang Mian 1
King King Ke ke 1543 Trice Stoneville 2B Uganda King King Lambright GL-5 Micnarie 210
37 86-1 Huanghe Henan Stoneville 4
736 遗传学报 Acta Genetica Sinica Vol.33 No.8 2006
and formulae used are shown below:
(1) Simpson diversity index, also known as Polymorphism Information Content: PIC = 1-ΣP i 2, where Pi represents the variation in frequency of the ith allele.
(2) Shannon-weaver diversity index, also re-ferred to as genotype diversity (H’): H’ = -ΣPiLnPi , where Pi indicates the variation frequency of the ith allele [9].
(3) Ne is the effective number of alleles for each locus: Ne = 1/∑Pi.
(4) Genetic similarity coefficients (Jaccard’s co-efficients) among varieties were calculated using the Qualitative Date program of NTSYSpc 2.1 software (Biostatistics Inc., New York, USA).
(5) Clustering analyses were performed using NTSYS-pc (version 2.11a) to calculate the genetic similarity matrices, and the dendrograms were con-structed by the unweighted pair-group method of arithmetic averages (UPGMA).
allele were BNL530, BNL3031, NBL1672, BNL2590, and BNL3259 having 8, 7, 6, 6, and 6 alleles, respec-tively. These primers possessed a strong potential for germplasm evaluation. Primers BNL3254 and TMB04 had only one polymorphic locus with less distinguishing potential among germplasms. 2. 2 Genetic similarity and clustering analyses 2. 2. 1 Source germplasm
A similarity matrix was built using genetic simi-larity between all possible combinations of two germplasms. Genetic similarity was calculated with NTSYS 2.1 using the Jaccard coefficient according to 0-1 data from the SSR markers. The genetic similar-ity coefficients among source germplasm ranged from 0.409 to 0.865, with an average of 0.610. The largest similarity was 0.865 between Stoneville 4 and Stone-ville 2B, which became source germplasm in China after they were introduced from Stoneville, USA. The similarity between Jiangsu mian 2 and DPL15 was 0.841. Jiangsu mian 2 had consanguinity with DPL14 and DPL15 as it was bred from a cross between DPL14 offspring and DPL15. Also, Uganda 4 had high similarity with Zhong mian suo 12 (0.816) and Zhong mian suo 7 (0.818). The common origin of these three varieties, which were derived from proge-nies of Uganda, had been validated by the SSR marker analysis. The lowest similarities were be-tween Foster 6 and Jin mian 2 (0.409), Jin mian 2 and DPL14 (0.413), Jin mian 2 and DPL15 (0.423), DPL15 and Lambrigh GL-5 (0.425), and Jiangsu mian 2 and Lambrigh GL-5 (0.421). The lower simi-larities between these varieties indicated that they were more distant from each other. In summary, the genetic similarity between source germplasms was lower, indicating that there were differences among the source germplasms. The low genetic similarity also showed that the source germplasms selected for analysis were representative of a range of cotton germplasm.
The cotton source germplasm could be divided into five groups on the basis of the average similarity coefficient (0.610) among the source germplasm (Fig. 1). The first group contained DPL14, DPL15, Uganda,
2 Results
2. 1 SSR marker multiple analyses
One hundred and thirty alleles with 80% poly-morphism among 43 source germplasms were de-tected. The average number of alleles for each SSR locus was 3.6 (Table 2). The effective number of al-leles for each SSR locus ranged from 1.385 to 7.405, with an average of 3.066. The PIC value for the SSR loci ranged from 0.278 to 0.865, with an average of 0.62. The genotype diversity was 0.451 to 2.039, with an average of 1.102. A total of 157 unique genotypes were detected by 36 pairs of polymorphic primers from a combination of 43 source germplasms. The average number of unique genotypes for each locus was 4.36. The polymorphic loci were distributed on cotton chromosomes 3, 4, 5, 8, 9, 10, 16, 18, 20, and 23, indicating that the variations of SSR alleles were dispersed in the whole cotton genome.
Among the source germplasm, two to eight SSR loci were obtained from each primer pair that was analyzed. The primers that amplified more than one
CHEN Guang et al.: Genetic Diversity of Source Germplasm of Upland Cotton in China as Determined by SSR Marker Analysis 737
Table 2 Allelic variation of 36 SSR loci among 43 source germplasms
Primer
No. of No. of polymorphic Polymorphism
alleles alleles (%)
8 3 3 2 2 2 2 4 5 3 2 4 2 2 6 1 2 5 4 3 4 1 2 2 2 2 3 2 2 2 3 2 4 1 5 2 2.889
1 0.75 0.75 1 0.667 0.667 1 1 0.833 0.75 0.667 0.667 0.667 1 0.857 0.5 1 0.833 0.8 0.75 0.8 0.333 0.667 1 0.667 1 0.75 0.667 1 1 1 0.667 1 0.333 1 0.667
No. of
genotypes
Chromosome
4 5 20
PIC H’
Ne
BNL530 8 BNL830 4 BNL1053 4 BNL1231 2 BNL1317 3 BNL1414 3 BNL1421 2 BNL1495 4 BNL1672 6 BNL1694 4 BNL2449 3 BNL2590 6 BNL2634 3 BNL2960 2 BNL3031 7 BNL3254 2 BNL3255 2 BNL3259 6 BNL3383 5 BNL3442 4 BNL3474 5 BNL3482 3 BNL3806 3 BNL3948 2 BNL3976 3 BNL4030 2 JESPR65 4 JESPR101 3 JESPR114 2 JESPR152 2 TMG10 3 TMK19 3 TMP02 4 TMB04 3 TME12 5 TMH08 3 Average 3.6
12 5 4 3 3 3 3 11 6 5 3 4 4 3 7 2 3 7 6 5 5 2 3 3 3 3 5 3 3 3 4 3 6 2 7 3 4.361
0.865 2.039 7.405 0.673 1.227 3.056 0.397 0.586 1.658
- 0.696 1.266 3.285
9＆23 0.614 1.008 2.588 9＆23 0.635 1.054 2.742 10 10 9 16 10 9
0.489 0.682 1.957 0.722 1.327 3.600 0.800 1.680 4.990 0.682 1.240 3.141 0.622 1.027 2.647 0.784 1.607 4.634
- 0.590 0.961 2.439 - 0.310 0.488 1.449 9＆23 0.834 1.867 6.019 18 10 3 23 18 26 25 20 5 22
0.341 0.525 1.518 0.436 0.628 1.774 0.780 1.588 4.536 0.718 1.358 3.550 0.737 1.360 3.804 0.778 1.558 4.512 0.602 0.989 2.513 0.477 0.670 1.911 0.551 0.873 2.229 0.496 0.689 1.982
- 0.665 1.096 2.986
- 0.681 1.229 3.139 - 0.590 0.962 2.441 - 0.499 0.692 1.996 - 0.278 0.451 1.385 - 0.593 0.967 2.458 - 0.605 1.000 2.535 - 0.708 1.305 3.422 - 0.651 1.074 2.868 - 0.779 1.548 4.523 -
0.625 1.037 2.669 0.620 1.102 3.066
Note: PIC = polymorphic information content, H’ = Shannon-weaver diversity index, Ne = effective number of alleles.
738 遗传学报 Acta Genetica Sinica Vol.33 No.8 2006
and the source germplasms derived from them. The
second group was mainly composed of Stoneville and their derived source germplasms. Lambright GL-5 and Micnarie 210 with very low gossypol content also belonged to this group. The third group included three source germplasms: Foster 6, Trice, and Ji mian 1. Jian mian 2 and Delfos531 formed the fourth and fifth group, respectively.
2. 2. 2 The source germplasm from different breed-ing periods
There were 11 source germplasms (Table 3) from an early breeding period, most of which were intro-duced from the USA except Guan nong 1. According to the similarity matrix, Stoneville 2B and Stoneville 4 had the greatest similarity (0.865), whereas DPL15 and Stoneville 4 had the least similarity (0.446). The average similarity coefficient of these source germ-plasms was 0.587. Guan nong 1 was derived from King of USA, and was an early maturity variety bred in 1930 in China. It was popular in the early maturity cotton growing area of northern China, including the Liaoning Province, and had become a source germ-plasm of the early period. More than 100 varieties were derived from Guan nong 1. According to the
Fig. 1 Dendrogram of 43 source germplasms based on SSR similarity coefficient
Table 3 SSR genetic similarity coefficient of source germplasms from different breeding periods
Breeding period First period (before the 1950s)
Similarity coefficient
0.587
Range
No. of samples
0.446-0.865 11
Second period (1950s-1960s) 0.630 0.445-0.818 25 Third period (1970s-1980s) 0.630 0.525-0.782 7
Overall 0.610 0.409-0.865 43
CHEN Guang et al.: Genetic Diversity of Source Germplasm of Upland Cotton in China as Determined by SSR Marker Analysis 739
Fig. 2 Dendrogram of first period source germplasm originating before the 1950s based on SSR similarity coefficient
similarity coefficient, Guan nong 1 has a close rela-tionship with DPL cotton. The early breeding period source germplasms were classified into three groups by the average similarity coefficient (0.578) (Fig. 2). The first group was DPL type including DPL15, DPL14, Guan nong 1, Coker 100, and empire cotton. The second group was Stoneville type containing Stoneville 2B, Stoneville 4, Stoneville 4B, Foster 6, and Trice. Delfos531 itself became the third group. Tracking the breeding history of these three groups, the ancestors of Delfos, DPL, and Trice had some relationship with Foster cotton. Foster with some specific characters was bred by the Foster farm, in USA, in 1904. This common ancestor shows that the genetic background of cotton was very narrow.
The second period source germplasm included 25 germplasms, 22 of which were derived from nine of the first-period source germplasms. The sources of the other three germplasms were Ke ke 1543, Zhong mian suo 7, and Uganda 4. The average similarity coefficient of these 25 source germplasms was 0.630. The similarity between Uganda 3 and Zhong mian suo 7 was the greatest (0.818). The lowest similarity was between Jin mian 2 and Jiangsu mian 2 (0.445), which were derived from Guan nong 1 and DPL14, respectively. The second period source germplasms were divided into three groups on the basis of the average similarity coefficient (0.63) (Fig. 3). The first group included varieties derived from DPL, Uganda 4, Zhong mian suo 7, 52-128, and Guan nong 1. The second group contained Foster offspring (Gan mian 1 and Shan mian 3), a DPL15-derived line (57-681), and a Stoneville-derived line (Liao mian 3). The third group contained four source germplasms, Xu zhou 1818, Yi shu hong, Chao yang 1, and Jin mian 2.
The third-period source germplasm included seven varieties, and their average similarity coeffi-cient was 0.630. The two closest basic germplasms were Ji mian 1 and Zhong mian suo 12 with a simi-larity coefficient of 0.728. The two farthest basic germplasms were Zhong mian suo 12 and Lambright GL-5 (0.525). Zhong mian suo 12 was developed from the cross of Uganda 4 and Ji mian 1. Zhong mian suo 12 and Ji mian 1 were classified into one
740 遗传学报 Acta Genetica Sinica Vol.33 No.8 2006
Fig. 3 Dendrogram of second period source germplasm originating between 1951 and 1970 based on SSR similarity coeffi-cient
group by SSR cluster analysis, indicating that the
derived variety had a close relationship with its par-ents at the DNA level.
The third-period source germplasms were classi-fied into two groups by average similarity coefficient (0.63) (Fig. 4). The first group was Lambright GL-5 and Micnarie 210 with lower gossypol content and 86-1 with resistance to the Fusarium wilt. The second group contained Ji mian 1, Zhong mian suo 12, and two early maturity cottons, Hei shan mian 1 and Zhong mian suo 10, which were derived from Guan nong 1. The comparison of SSR genetic similarity coefficients of different breeding periods is shown in Table 3. The average similarity coefficient of the first-period source germplasm was the lowest (0.587) with a broad range (0.446-0.865), indicating that this germplasm had many differences at the genome level. The similarity coefficients of the second- and the third-period source germplasms were greater than that of the first period, and the range was also smaller. This indicates that the similarity in the second- and the third-period source germplasms increased. The main
reason was that most of the source germplasms of the second and the third periods were derived from those of the first period source germplasms. Moreover, some genetic differences among the varieties were lost in the process of selection of high-quality and high-yield traits in the breeding program, and this resulted in the genetic base for breeding becoming narrower.
2. 2. 3 Source germplasm from different cotton
growing areas
From the similarity coefficient of source germplasms from different cotton areas (Table 4), we found the fol-lowing results. The similarity among the introduced American varieties was the lowest, which indicated sig-nificant genetic differences among the American varieties. The similarity among the source germplasm from the Huanghe River valley cotton area was the highest. The similarity among the source germplasm of Yangtze River valley area was also high, as their average similarity coef-ficient reached 0.610. These coefficients showed that the variation of source germplasms in the two main cot-ton-growing areas of China declined compared with that of the germplasms introduced originally. However, the
CHEN Guang et al.: Genetic Diversity of Source Germplasm of Upland Cotton in China as Determined by SSR Marker Analysis 741
Fig. 4 Dendrogram of third period source germplasm originating between 1971 and 1990 based on SSR similarity coeffi-cient
that of the introduced germplasm (0.585, Table 5).
This indicated that it would be difficult for the ge-maturity cotton area was lower than that in the Huang-he
netic base of domestic germplasms to exceed foreign and Yangtze River areas. The breeders usually focused on
using the varieties with high quality, high yield, and germplasms, which could be the result of the limited
genetic resources used in China. stronger adaptability, and this likely made the genetic
It was necessary to analyze the introduced source background among the varieties in the Huang-he and
germplasm as the source germplasms in China were all Yangtze River areas narrower.
derived from introduced varieties. There were four 2. 2. 4 The domestic and introduced source germ-groups of foreign source germplasms measured with the plasm
average similarity coefficient of source germplasm. The Many of the Chinese breeding source germplasms
first group was composed of DPL type, Empire, had been based on the introduction, selection, and do-Coker100, and Uganda cotton. The second group was mestication of germplasms from other countries. As a
composed of Stoneville type, Foster, and Trice. The result, the average genetic similarity coef ficient of
third group was composed of those with lower gossypol domestic source germplasm (0.624) was higher than
content and Ke ke 1543 introduced from Russia. The
Delfos cotton formed a group by itself (Fig. 5). Table 4 SSR genetic similarity coefficient of source
germplasm from different cotton growing areas
SSR genetic similarity coefficient of domestic and Cotton-growing areas Similarity coefficient No. of samples Table 5 introduced germplasm Yangtze River areas 0.610 10
Huanghe areas 0.651 11 Source germplasmSimilarity coefficient No. of samples Northern China 0.586 6 Domestic 0.624 27
American 0.576 14 similarity among the source germplasm in the north early
Overall 0.610 43
Introduced 0.585
16
742 遗传学报 Acta Genetica Sinica Vol.33 No.8 2006
Fig. 5 Dendrogram of introduced source germplasm based on SSR similarity coefficient
3 Discussion
The genetic relationships among source germ-plasms of Upland cotton in China have been analyzed
on the basis of SSR molecular markers. The main results are summarized below:
1. The average genetic similarity coefficient of source germplasms was 0.610 and ranged from 0.409 to 0.865. This suggested that the analyzed source germplasms possessed vast diversity, a large extent of variation, and were a general representative of the diversity of source germplasms.
2. The average genetic diversity coefficients of the SSR markers for the source Upland germplasms of the first, second, and third breeding periods were 0.587, 0.630, and 0.630, respectively. This implied that the genomic difference among the modern source germplasm had gradually decreased compared to that of the early source germplasm. This was probably a result of the narrow breeding base as the breeders used a limited number of varieties with high quality and high yield.
3. The similarity coefficients of the SSR markers
among the source germplasms in different Chinese cotton growing areas were different. The diversity of the source germplasm in the northern early maturity cotton area was largely preserved. However, the di-versity declined distinctly in the Huanghe and Yang-tze River areas compared to the diversity of the in-troduced source germplasm. The varieties with high quality, high yield, and stronger adaptability were preserved, whereas those with poor adaptability were eliminated through selection, and this lessened the genetic difference among the varieties in Huanghe and Yangtze River areas.
4. The SSR similarity coefficient of domestic source germplasm (0.624) was higher than that of the introduced germplasm (0.585). This suggests that the introduced source germplasm had adapted to the en-vironment and climate in China, and many varieties were derived from them. However, the limited ge-netic diversity of domestic source germplasm could never exceed the diversity of the introduced germ-plasms.
China is not a native cotton growing area; there-fore, its cotton breeding and production was based on
CHEN Guang et al.: Genetic Diversity of Source Germplasm of Upland Cotton in China as Determined by SSR Marker Analysis 743
the introduced germplasms. Source germplasm is the foundation of cotton breeding, the basis of derived varieties, and plays an important role in the cotton production in China. Therefore, it is necessary to study the source germplasm. The genetic diversity of 43 source germplasms of Upland cotton, classified according to the quantity of derived varieties and their pedigrees, have been assessed in this study. The large differences among the source germplasms cov-ered 95% of the diversity of varieties bred in China and proved that the source germplasms selected were a representative sample of Chinese cotton [2].
The source germplasms contributed considerably to breeding programs in China, although their types and amounts were limited. This increased the similar-ity among their derived varieties, and made the ge-netic base to narrower. This is explained by the fol-lowing experimental results: the genetic diversity of the source germplasm of the second and the third breeding period was lower than that of the first period; the diversity of domestic source germplasm declined compared to that of the introduced germplasm; and the diversity among the derived varieties decreased compared to those of their source parents. The main reasons were that the source germplasms of the latter two breeding periods were mostly derived from the first period and the domestic source germplasms were mostly derived from introduced germplasm sources. Other research[10-14] on cotton’s genetic diversity us-ing RAPD, ISSR, and SSR markers also showed that the genetic base of cotton breeding was narrow in China. For instance, Bie et al.[10] clustered 30 varie-ties from three main cotton-growing areas in China into three groups and showed that their genetic base was narrow. Their diversity also had some relation-ship with their pedigrees on the basis of RAPD marker and phenotype analyses of the varieties. Xu et al.[15] reported that the genetic diversity of the varie-ties bred by CRICAAS from the end of the 1970s to the middle of the 1990s was higher than that of the varieties from the Hebei Province. The 19 varieties bred in the Hebei Province also had a much narrower base. Xu et al.[16] analyzed the genetic diversity of the varieties resistant to Fusarium wilt using RAPD markers. This research pointed out that the genetic diversity of Upland cotton was lower in China, but it was vital for introducing Fusarium wilt resistance genes from G. arboreum, G. barbadense, and other species into Chinese G. hirsutum. Wu et al.[17] studied 36 domestic and introduced cultivars of Upland cot-ton with SSR markers and morphological characters and showed that there were some genetic differences among these materials, but the genetic diversity of these cotton cultivars was low. This again suggested that the genetic base of Upland cotton was narrow. When Xu et al.[18] compared Upland cotton in Yang-tze River and Huang-he River areas by RAPD marker analysis, the genetic diversity of the varieties in both areas were similar. This further supported the view that common germplasm sources and same breeding targets, similar breeding methods, and policies could be the most important factors to influence the genetic diversity of these two cotton-growing areas in China.
Molecular markers can show genetic diversity at the genome level, whereas the phenotypes express the interactions of genes and environments. The majority of domestic source germplasm and their derived va-rieties have been bred in China by selecting and do-mesticating introduced varieties. The domestic varie-ties have higher yield and improved quality, but the selection pressure and limited source germplasm has narrowed the genetic base in domestic cultivars.
The development of a core collection, which identifies a small number of unique germplasms to represent the genetic diversity present in a larger col-lection of germplasm, is a very important research area. On the basis of this study, it is obvious that source germplasms are the most diverse part of the cotton collection in China. This research of source germplasms by SSR marker analyses will provide important methods and technologies for the construc-tion of a Chinese cotton core collection. It will also provide data for variety improvement, and updating and enhancing the diversity of germplasms. Further-more, both source germplasms and their derived va-rieties are studied systematically to understand the genetic base of cotton breeding more clearly in our program.
744 遗传学报 Acta Genetica Sinica Vol.33 No.8 2006
Acknowledgments: We thank Prof. Liu Guo-Qiang
and deputy Prof. Sun Junling, who provided valuable suggestions for the paper , and helped review this article. We also acknowledge Dr. Lori Hinze, USDA-ARS, College Station, TX 77845, for a critical review of this article. References:
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CHEN Guang et al.: Genetic Diversity of Source Germplasm of Upland Cotton in China as Determined by SSR Marker Analysis 745
我国陆地棉基础种质遗传多样性的SSR分子标记分析
陈 光，杜雄明
中国农业科学院棉花研究所/农业部棉花遗传改良重点开放实验室，安阳 455004
摘 要：利用398对BNL、JESPR 、TMB等SSR引物，对不同亲本来源、不同选育时期、不同种植生态区的43份陆地棉基础种质进行了遗传多样性的SSR分子标记分析。扩增产物用8%的非变性聚丙烯酰胺凝胶检测,银染观察并照相。遗传多样性带型分析按位点多态信息量（PIC），Shannon-weaver多样性指数（H’）等方法，利用NTSYSpc2.1软件计算品种间的遗传相似系数（Jaccard系数），并用类平均法（UPGMA）进行聚类。结果表明所选择多态性引物分布在棉花基因组的第3、4、5、8、9、10、16、18、20、23号等染色体上，36对多态性引物在基础种质中扩增等位基因130个，其中多态性等位基因占80%,每个引物扩增等位基因2～8个，平均3.6个，PIC为0.278～0.865，平均0.62，基因型多样性（H’）为0.451～2.039，平均1.102，基础种质间SSR遗传相似系数平均为0.610，变幅为0.409～0.865，这说明所选基础种质基因组水平的多样性较丰富，变化范围大、代表性强。按品种不同选育时期来讲，第一、二、三期基础种质的SSR分子标记平均遗传相似系数分别是0.587、0.630、0.630，说明现代基础种质比早期基础种质在基因组水平的差异呈下降的趋势，可能是由于育种者偏重于使用优质高产性状的亲本品种，致使我国棉花的育种基础逐渐变窄。不同棉区基础种质SSR标记性状差异大，北部特早熟棉区基础种质间的SSR标记的多样性大于黄河、长江棉区，主要原因是长江、黄河棉区的育种过分强调高产、优质品种选育，品种间的差异变小；基础种质中的国内品种SSR相似系数（0.624）比引进品种（0.585）高，说明国内品种在遗传多样性上目前还没有超越国外品种。总之，我国棉花现代基础种质比早期基础种质的遗传多样性呈下降的趋势，黄河、长江主产棉区基础种质的遗传多样性还没有超过国外基础种质，品种间的遗传背景较为狭窄，还必须采用多种途径丰富我国棉花种质资源的遗传多样性。 关键词：基础种质；SSR；遗传多样性
作者简介：陈光（1978-），女，硕士研究生，专业方向：生物化学与分子生物学。E-mail：cgcjca637@sohu.com
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