Min and Max, the minimum value and maximum value in the sample; Mean, the average value of the sample; CV (%), coefficient of variation; F, ratio of MSA/MSE; P, P value; n, sample size.
Comparison of morphological measurements (a), pseudobulb weight (b) and the main active ingredient content (c) in different growth stages and different species of B. striata and B. ochracea. Values are the mean ± SD (standard deviation), and different lowercase letters indicate significant differences with the parameter of P < 0.05.
There were significant differences in pseudobulb weight among different species and different populations, and the coefficient of variation (CV) of pseudobulb weight was higher than the CVs of other phenotypic traits (Fig. 2, Table 1). In detail, the weight of the one-year-old pseudobulb of different populations varied from 5.02 to 86.08 (g), and the CV (%) was 69.55. The GXHZ, HBXG, HBLT, HBMC and HBWH populations were ranked as the top five, while the last five populations were HBXS, HBSY, HBWF (B. ochracea), SXXX, and GXLY. For the two-year-old pseudobulb, the weight in different populations varied from 12.58 to 176.35 (g), and the CV (%) was 66.25. GXHZ, HBXG, HBLT, AHBZ and GZZY were ranked as the top five populations, while the last five populations were SCLS, HBSY, HBWF (B. ochracea), SXXX and GXLY. For the three-year-old pseudobulb, the weight in different populations varied from 23.13 to 329.42 (g), and the CV (%) was 72.34. GXHZ, AHBZ, HBXG, HBLT and HBSN were ranked as the top five populations, while the last five populations were SYZS, HBSY, HBWF (B. ochracea), SXXX, and GXLY. For the four-year-old pseudobulb, the weight in the different populations varied from 31.63 to 442.77 (g), and the CV (%) was 71.06. HBLT, AHBZ, HBWH, HBSN and HBXG were ranked as the top five populations, while the HBWF (B. ochracea), HBSY, SXXX, HNCL and GXLY populations showed lower weights. In general, except in GXHZ, HNCL and SCWY, the weight of the pseudobulbs increased with the growing period, while the average annual growth rates of the pseudobulb were 2.42, 1.69 and 1.31, respectively, showing a decreasing trend. There was a significant difference between the two species, and the average one-, two-, three- and four-year-old pseudobulb weights of B. ochracea were 19.66, 63.26, 102.07 and 140.95 (g), which were lower than the 37.44, 85.72, 144.79 and 193.49 (g) of B. striata, respectively (Table 2, Fig. 2a).
Table 2 The fresh weight of pseudobulbs of 28 populations of B. striata and 5 populations of B. ochracea
No.
|
Population code
|
1-year-old (2017)
|
2-year-old (2018)
|
3-year-old (2019)
|
4-year-old (2020)
|
1
|
GXHZ
|
86.08±13.29a
|
176.35±78.70a
|
329.42±219.32a
|
285.73±177.13c
|
2
|
GXLY
|
5.02±3.07k
|
12.58±7.58n
|
23.13±13.11i
|
31.6316.53m
|
3
|
YNBS
|
18.45±11.58hij
|
52.16±10.50jklmn
|
100.95±36.55ghi
|
167.60±40.73efghi
|
4
|
GZZY
|
59.77±28.91c
|
152.43±32.84abc
|
253.88±20.81bc
|
283.08±34.85cd
|
5
|
HNLS
|
44.43±11.07d
|
98.21±30.68efghi
|
133.16±57.66efg
|
212.06±158.28def
|
6
|
HNSZ
|
15.55±5.97hij
|
67.42±51.50ijkl
|
131.45±71.33efgh
|
186.64±90.39defgh
|
7
|
HNCL
|
36.55±11.74def
|
68.55±19.58ijkl
|
66.52±27.34hi
|
36.89±20.47lm
|
8
|
YCWZ
|
39.67±19.23d
|
80.14±20.82ghij
|
199.36±64.48cd
|
254.91±79.04cde
|
9
|
SCLS
|
21.24±6.14ghi
|
36.07±16.49lmn
|
69.70±32.87hi
|
80.33±14.79ijklm
|
10
|
SCWY
|
26.66±11.81fgh
|
125.10±39.67bcde
|
108.91±32.24fgh
|
75.51±45.11ijklm
|
11
|
HBYL
|
37.56±6.41de
|
78.95±21.01ghij
|
141.17±34.41defg
|
194.37±72.01defg
|
12
|
HBLC
|
39.45±13.33d
|
103.59±40.97defgh
|
136.05±36.46efg
|
153.30±45.66fghi
|
13
|
HBXE
|
24.48±5.80gh
|
73.74±44.13hijk
|
94.49±36.70ghi
|
110.01±58.34ijklm
|
14
|
SYZS
|
24.41±6.79gh
|
45.02±28.37klmn
|
61.25±41.19hi
|
97.80±25.16ijklm
|
15
|
HBHF
|
39.79±16.95d
|
68.82±41.16ijkl
|
132.21±63.03efg
|
213.68±92.48def
|
16
|
HBXS
|
15.24±4.52hijk
|
62.84±28.88jklm
|
93.2755.52ghi
|
130.48±54.36fghij
|
17
|
HBJS
|
30.10±11.40efg
|
57.99±40.33jklm
|
94.61±38.39ghi
|
152.47±50.05fghi
|
18
|
HBSN
|
36.23±6.81def
|
131.69±27.26bcd
|
263.91±68.91b
|
367.12±89.39ab
|
19
|
HBWH
|
64.41±20.01c
|
119.97±15.68bcdef
|
193.36±90.63cde
|
399.53±116.89ab
|
20
|
XSSC
|
15.73±9.21hij
|
53.82±11.89jklm
|
80.64±8.71ghi
|
110.45±33.88hijklm
|
21
|
HBJM
|
18.84±2.45hij
|
48.38±12.00jklmn
|
99.00±21.87ghi
|
117.80±37.57hijkl
|
22
|
HBXG
|
84.92±15.84a
|
162.78±55.11ab
|
299.58±33.65ab
|
333.50±49.51bc
|
23
|
HBMC
|
68.03±17.15bc
|
109.06±38.18cdefg
|
185.71±46.99cde
|
254.60±103.59cde
|
24
|
HBLT
|
77.35±14.76ab
|
158.96±39.57ab
|
286.61±44.59ab
|
442.77±78.20a
|
25
|
HBSY
|
13.47±3.62ijk
|
28.49±10.05mn
|
40.58±17.27i
|
62.82±30.89jklm
|
26
|
AHBZ
|
46.18±19.93d
|
152.93±59.94abc
|
305.60±58.49ab
|
401.42±97.18ab
|
27
|
JSNJ
|
42.08±16.98d
|
121.91±41.78bcdef
|
182.00±69.76de
|
210.09±80.87def
|
28
|
SXXX
|
9.20±2.33jk
|
15.72±13.22n
|
36.14±22.81i
|
38.29±31.72klm
|
29
|
HNLS
|
20.32±7.00hi
|
94.38±53.56fghi
|
163.38±81.45def
|
199.00±111.81def
|
30
|
HNSZ
|
15.68±6.39hij
|
45.89±25.53klmn
|
91.74±17.76ghi
|
128.95±74.95fghijk
|
31
|
HBWF
|
9.81±6.42jk
|
17.78±7.92mn
|
38.68±26.01i
|
53.75±35.43jklm
|
32
|
SCLS
|
28.86±7.33fg
|
73.47±36.30jk
|
87.41±56.11ghi
|
124.77±85.79ghijk
|
33
|
SXZP
|
21.74±4.66ghi
|
55.81±18.35jklm
|
110.90±13.73fgh
|
158.60±22.78fghi
|
No. 1-28 are B. striata, No. 29-33 are B. ochracea. Red represents the top five populations of pseudobulb weight in different years, while green represents the last five populations. Values represent the mean ± SD of ten individuals, and different lowercase letters with superscripts indicate significant differences between populations with the parameter of P < 0.05.
Determination of active ingredients content
The polysaccharide, total phenol and militarine contents of pseudobulbs were determined among different species and populations. The results showed that significant differences were observed among different populations, with a minimum F value of 284.97 and P values much lower than 0.05 (Table 1). Overall, there were no differences in the content of the main active ingredients among different species and different planting years (Fig. 2c).
Most populations had an excellent phenotype in terms of polysaccharide content, which varied from 8.52 to 59.77 (%). 8, 12, 15 and 13 populations accounted for more than 50% of the one-, two-, three- and four-year-old pseudobulbs, and 17, 12, 13 and 9 populations ranged from 40% to 50%, respectively (Table 3). The average polysaccharide contents of one-, two-, three- and four-year-old pseudobulbs of B. ochracea were 45.18, 47.57, 44.70 and 40.45 (%), and the average polysaccharide contents of one-, two-, three- and four-year-old pseudobulbs of B. striata were 45.00, 45.32, 47.34 and 42.92 (%), which means the content of polysaccharide in pseudobulb of B. striata was three-year-old > two-year-old > one-year-old > four-year-old, while which of B. ochracea was two-year-old > one-year-old > three-year-old > four-year-old (Fig. 2c). In general, the top five populations with the highest polysaccharide content in the pseudobulbs of the average of four years were HBYL, SXZP (B. ochracea), AHBZ, HBHF and HBWF (B. ochracea), while the last five populations were GXLY, HNLS (B. ochracea), HBJM, HNSZ (B. ochracea) and YNBS.
Obvious variation was observed in the content of total phenol, which varied from 0.90 to 5.00 (%). There were 14, 11, 16 and 13 populations that were higher than 4% of the one-, two-, three- and four-year-old pseudobulbs, and 12, 15, 12 and 12 populations ranged from 3% to 4%, respectively (Table 3). The average total phenol content of one-, two-, three- and four-year-old pseudobulbs of B. ochracea were 3.96, 3.57, 3.89 and 3.35 (%), which were lower than the 3.98, 4.14, 4.33 and 3.43 of B. striata, respectively. Therefore, the order of the contents of total phenol in the pseudobulb of B. striata was three-year-old > two-year-old > one-year-old > four-year-old, while that in B. ochracea was one-year-old > three-year-old > two-year-old > four-year-old (Fig. 2c). In general, the top five populations with the highest average total phenol contents in pseudobulbs of four years were JSNJ, HBYL, HBXG, SCWY and HBLT, while the last five populations were YCWZ, HNLS (B. ochracea), SXXX, YNBS and HBJM.
Table 3 Active ingredient contents of 28 populations of B. striata and 5 populations of B. ochracea (listed at the end of the document)
The average milliarine contents of the one-, two-, three- and four-year-old pseudobulbs of B. ochracea were 1.43, 1.62, 1.85 and 1.64 (%), which were generally higher than those of B. striata (1.44, 1.39, 1.45 and 1.60 (%), respectively). That is, the order of the militarine contents of the pseudobulbs of B. striata was four-year-old > three-year-old > one-year-old > two-year-old, while that in B. ochracea was three-year-old > four-year-old > two-year-old > one-year-old (Fig. 2c). Militarine contents varied from 0.51 to 4.12 (%), 4, 4, 6 and 5 populations accounted for more than 2% of the one-, two-, three- and four-year-old pseudobulbs, and 7, 5, 9 and 12 populations accounted for 1.5% to 2%, respectively (Table 3). Overall, the top five populations with the highest average militarine contents of the pseudobulbs of four years were HNSZ, SCLS (B. ochracea), HBWF (B. ochracea), YCWZ and SXXX, while the HBHF, HNCL, HBMC, HBXG and SXZP (B. ochracea) populations were the five populations with the lowest contents.
GRA of the pseudobulb weight, pseudobulb growth ratio and main active ingredient content showed that there are 2 populations greater than 0.6 and 15 populations greater than 0.5. HNSZ, AHBZ, HBLT, HBSN and JSNJ were ranked as the top five, indicating that these populations have good characteristics in comprehensive yield and quality, while the last five populations were SXXX, SYZS, GXLY, HBJM and YNBS (Table 4).
Table 4 Correlation degree and rank of pseudobulb weight and main active ingredient contents based on gray relational analysis
Population code
|
Correlation degree
|
Relative correlation degree
|
Rank
|
relative to optimal seq.
|
relative to least seq.
|
HNSZ
|
0.721
|
0.432
|
0.625
|
1
|
AHBZ
|
0.683
|
0.434
|
0.611
|
2
|
HBLT
|
0.676
|
0.471
|
0.589
|
3
|
HBSN
|
0.631
|
0.452
|
0.583
|
4
|
JSNJ
|
0.628
|
0.465
|
0.574
|
5
|
GXHZ
|
0.635
|
0.501
|
0.559
|
6
|
HBWH
|
0.611
|
0.483
|
0.558
|
7
|
SCLS (B. ochracea)
|
0.607
|
0.485
|
0.556
|
8
|
HBXG
|
0.649
|
0.525
|
0.553
|
9
|
HBYL
|
0.639
|
0.532
|
0.545
|
10
|
SCWY
|
0.608
|
0.508
|
0.545
|
11
|
GZZY
|
0.566
|
0.505
|
0.528
|
12
|
HBXS
|
0.577
|
0.522
|
0.525
|
13
|
SXZP (B. ochracea)
|
0.595
|
0.558
|
0.516
|
14
|
HNLS
|
0.552
|
0.538
|
0.506
|
15
|
HBLC
|
0.528
|
0.518
|
0.505
|
16
|
HBHF
|
0.571
|
0.568
|
0.501
|
17
|
HBWF (B. ochracea)
|
0.574
|
0.575
|
0.500
|
18
|
XSSC
|
0.525
|
0.533
|
0.496
|
19
|
YCWZ
|
0.488
|
0.531
|
0.479
|
20
|
HBMC
|
0.531
|
0.590
|
0.474
|
21
|
HNLS (B. ochracea)
|
0.501
|
0.571
|
0.467
|
22
|
SCLS
|
0.515
|
0.597
|
0.463
|
23
|
HBXE
|
0.476
|
0.593
|
0.445
|
24
|
HNSZ (B. ochracea)
|
0.467
|
0.599
|
0.438
|
25
|
HNCL
|
0.494
|
0.652
|
0.431
|
26
|
HBJS
|
0.451
|
0.612
|
0.424
|
27
|
HBSY
|
0.445
|
0.633
|
0.413
|
28
|
SXXX
|
0.446
|
0.655
|
0.405
|
29
|
SYZS
|
0.438
|
0.646
|
0.404
|
30
|
GXLY
|
0.440
|
0.665
|
0.398
|
31
|
HBJM
|
0.406
|
0.694
|
0.369
|
32
|
YNBS
|
0.409
|
0.757
|
0.351
|
33
|
The populations of unmarked species are B. striata.
Correlation analysis of phenotypic traits, pseudobulb weight and active ingredient content
To investigate which phenotypic traits may determine the yield of pseudobulb and active ingredient content, and whether there is a correlation between the yield of pseudobulb and active ingredient content, Pearson correlation analysis was performed for two-, three- and four-year-old plants among 13 indexes, including 9 morphological indexes of aboveground parts, pseudobulb weight indexes and 3 active ingredient indexes. The results showed that except for the negative correlation between the militarine content and other indexes, the other 12 indexes were positively correlated (Fig. 3).
The correlation coefficient of plants in 2018 ranged from -0.401 to 0.861. The correlation coefficients between plant height and leaf blade length, and between inflorescence height and inflorescence length were greater than 0.8 (0.806 and 0.861, respectively). There were 4 other correlation coefficients greater than 0.7, 3 correlation coefficients greater than 0.6, 5 correlation coefficients greater than 0.5, and 7 correlation coefficients greater than 0.4, and all of them showed extremely significant differences with P values of less than 0.01. Overall, there were significant correlations between phenotypic traits except leaf number, flower number and fruit number. There were significant correlations between pseudobulb weight and plant height, leaf blade width, stem diameter, and total phenol content. There were significant correlations between polysaccharide content and leaf blade width, stem diameter, and total phenol content. There were significant correlations between the total phenol content and leaf blade width, stem diameter, pseudobulb weight, and polysaccharide content. There was a significant negative correlation between militarine content and leaf blade length (Fig. 3a).
The correlation coefficient of plants in 2019 ranged from -0.282 to 0.920. The correlation coefficient of inflorescence height and inflorescence length was 0.920. The correlation coefficients of plant height with inflorescence height and inflorescence length were greater than 0.8. There were 6 other correlation coefficients greater than 0.7, 7 correlation coefficients greater than 0.6, and 7 correlation coefficients greater than 0.5, and all of them had extremely significant differences with a P value less than 0.01. Overall, there were significant correlations between phenotypic traits except leaf number, and fruit number. There were significant correlations between pseudobulb weight and all phenotypic traits except for fruit number; however, the three indexes with the strongest correlations were the same as those in 2018, which were plant height, leaf width and stem diameter. The correlation between polysaccharide content and phenotypic traits was lower than that in 2018, and the same trends were observed for total phenol and militerine. However, there was still a significant correlation between the polysaccharide and total phenol content (Fig. 3b).
The correlation coefficient of plants in 2020 ranged from -0.331 to 0.931, and the highest value was obtained for inflorescence height and inflorescence length. There were 2 other correlation coefficients greater than 0.8, 3 correlation coefficients greater than 0.7, 6 correlation coefficients greater than 0.6, and 6 correlation coefficients greater than 0.5, and all of them had extremely significant differences with a P value less than 0.01. Overall, there were significant correlations between phenotypic traits except leaf number and fruit number. There were significant correlations between pseudobulb weight and plant height, leaf blade width, leaf number, and stem diameter. There were significant correlations between polysaccharide content and leaf blade width, stem diameter, total phenol content. There were significant correlations between the total phenol content and leaf blade width, and polysaccharide content. There was also a nearly negative correlation between the militarine content and other indexes (Fig. 3c).