Whole-genome sequencing of Chinese native goat offers biological insights into cashmere fiber formation
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Abstract
Cashmere evolved naturally in the goat, and almost all breeds of goat can produce more or less cashmere fibers. However, the genetic alterations underlying cashmere trait selection are still unclear.We sequenced 120 Chinese native goat including two cashmere goat breeds (Ujumain, Chaidamu) and six ordinary goat breeds (Jining Gray, Matou, Guizhou Black, Jintang Black, Yunnan Black Bone, Chengdu Brown). The genome-wide selective sweep of cashmere goat and ordinary goat revealed a novel set of candidate genes as well as pathways, such as Nuclear factor kappa-B and Wnt Signaling pathways. Of them, the LHX2 gene regulating hair follicle development, was evident from the strongest selection signal when comparing the Uhumqin cashmere goat and ordinary goat. Interestingly, we identified a 582bp deletion at 367 kb upstream of LHX2 with higher frequency in cashmere goats and their ancient relatives. This mutation probably rises along the breeding procedures, and is putatively responsible for cashmere production and diameter, as revealed by association studies. Luciferase assay shows that the deletion, which acts as an insulator, restrains the expression of LHX2 by interfering its upstream enhancers.Our study discovers a novel insulator of the LHX2 involved in regulating cashmere production and diameter, which would be beneficial to understanding hair follicle development and regeneration. Our findings also provide new insights into the genetic formation of cashmere, and facilitate subsequent molecular breeding for cashmere goat improvement.
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Abstract
Reviewer1: Mahesh Neupane
Nicely written paper on selection signatures for Cashmere goats with detailed analysis and possible deletion. Here are some of the suggestions to improve the paper:
How was the optimal size of K determined in admixture? Please review the formatting on the manuscript, for example page 5 and page 6 figure have some formatting errors. Was sample size enough for all the comparison? What was the power of study design. How the results from mouse and human cell line justified for comparison with goat? Very good job of supplying all the codes used in the programs. Perhaps this codes or parameters can be combined together as supplemental material or GitHub repository.
Reviewer2: Yixue Li
The authors raised an interesting question, hoping to discover the genetic mechanisms associated with cashmere traits for …
Abstract
Reviewer1: Mahesh Neupane
Nicely written paper on selection signatures for Cashmere goats with detailed analysis and possible deletion. Here are some of the suggestions to improve the paper:
How was the optimal size of K determined in admixture? Please review the formatting on the manuscript, for example page 5 and page 6 figure have some formatting errors. Was sample size enough for all the comparison? What was the power of study design. How the results from mouse and human cell line justified for comparison with goat? Very good job of supplying all the codes used in the programs. Perhaps this codes or parameters can be combined together as supplemental material or GitHub repository.
Reviewer2: Yixue Li
The authors raised an interesting question, hoping to discover the genetic mechanisms associated with cashmere traits for breed improvement. The authors sequenced 120 native Chinese goats, including 2 cashmere goat breeds and 6 common goat breeds. Through analysis, the authors found and believe they confirmed that a 582 bp deletion at 367 kb upstream of LHX2 is involved in regulating cashmere yield and cashmere diameter. The results are very interesting, and if they convincingly answer the questions below, acceptance for publication is recommended. 120 goats, are they inbred, and are there enough to get a statistically significant result? What is the statistical basis? The article begins to describe: 582del and 504del are both correlated with cashmere yield, but only 582del is also significantly correlated with fiber diameter, while 504del has no significant correlation with fiber diameter. Then he added: The interaction effect between 582del and 504del was significantly correlated with cashmere fiber diameter, indicating an interaction between the two genes. What is the mechanism of this interaction? How they are significantly related to cashmere fiber diameter, further elaboration is needed. On the one hand, the text mentions that the deletion sequence 582del in the upstream region of the LHX2 gene may act as an insulator, preventing the function of the LHX2 enhancer. Later, it is mentioned that the deletion of the LHX2 insulator 582del increases the expression of LHX2 and promotes the growth of cashmere fibers during the growth period. There seems to be a contradiction here, please explain. To confirm the insulator function of the 582del sequence, the authors synthesized a 551 bp DNA fragment and inserted it into the pGL3 plasmid downstream and upstream of the SV40 promoter, and then co-transfected human 293T cells and mouse 3T3 cells, and thus confirmed the insulator function of the 582del sequence. Here: (1) the two sequences are not identical in length and identity, (2) using mouse and human cell lines respectively, how do we conclude that the 582del sequence will have the same function in goat? What is the experimental logic and biological logic here? Hopefully a convincing explanation can be given.
Reviewer3: Yu Jiang
This manuscript resequenced 42 cashmere goats and 78 ordinary goats, performing Genome-Wide Selective Sweeps and then a 582 bp deletion in the thirteenth intron region of DENND1A upstream of LHX2 was found to increase cashmere yield. This discovery provides resources for the development of the wool industry and the enrichment of animal genetic resources. However, the description in some parts of the manuscript is very rough, and many attachments are missing. Major concerns: 1.In the result "Plausible Causative Mutation near LHX2", you selected a lot of cashmere and fiber diameter data for association analysis and get Fig 5e, but your results may have high false positives. The author should demonstrate that the deletion is significantly associated with phenotype after excluding factors such as gender, age and so on. 2.It is found from Fig.2 a that MT and JNG contain a large number of cashmere goat pedigrees. When they are selected and analyzed with cashmere goat samples, the results may be affected by this mixing. The author should consider the particularity of these samples when using them. Perform subsequent analysis. 3.In Fig 3, are strongly selected loci linked to surrounding loci? Do these sites have an effect on gene expression? 4.Fig 3c & d, the horizontal and vertical coordinates of your two graphs are the same, but the trends of the graphs are different. Please mark clearly what the two graphs describe in the legend and the paper. Minor concerns: 5.In the abstract, "Luciferase assay shows that the deletion, which acts as an insulator, restrains the expression of LHX2 by interfering its upstream enhancers", but in the result, "Therefore, the deletion of the LHX2 insulator increases the expression of LHX2 and promotes cashmere fiber growth at the anagen stage, while deletion of the FGF5 enhancer reduces the expression of FGF5, inhibiting the regression". The conclusion is inconsistent, please clarify the logic of the paper and draw the correct conclusion. 6."Luciferase assay shows that the deletion, which acts as an insulator, restrains the expression of LHX2 by interfering its upstream enhancers. Our study discovers a novel insulator of the LHX2 involved in regulating cashmere production and diameter." These two sentences are easy to confuse us, and it will make people understand that two insulators are found on LHX2, one to suppress expression and one to regulate cashmere production and diameter, and it is recommended to modify. 7.There are inconsistencies in the sample names in the paper. For example,you use IRWG in the front of the sentence and IRW in the back,please unify the name. At the same time, the paper contains many spelling and symbol errors, such as "mddle", "goatswith", symbol repetition and so on. (1)In the STRUCTURE analysis, When K = 4, we observed five separate clusters: IRWG and ANG in west Asia, YNBB, GZB, JTB and CDB in southwest China; cashmere goat in north China; MT and JNG in mddle east China; and Korean goats in south Korea. At K = 6, goats in the southwest China further split into two geographic subgroups: the Yunnan-Kweichow Plateau group including YNBB and GZB goats, and the Chengdu Plain group including CDM and JTB goats. Two west Asian goats (IRW and ANG) were also separated . (2)More interestingly, we found that the 582del has a high frequency in the IRWG population (80.9 %), while the 504del was absent, which is also consistent with previous research. (3)10 JTB from Jintang County of Sichuan Province; 12 CDB from Chengdu City of Sichuan Province".." To further evaluate whether these two deletion variants were related to cashmere (4)traits, we selected 235 CDMC goatswith cashmere yield (Supplementary Fig. 22, Supplementary Table s8) and 581 CDMC goats with fiber diameter records (Supplementary Fig. 23, Supplementary Table s9) for association analysis. (5)Fig 2b, "KOG". 8."We inspected all variants within exons to identify the potential causal mutation around the DENND1A-LHX2 locus; however, no coding variants were found. " Please put the information of the relevant sites in the attachment, only one sentence will make the paper unconvincing. 9."Analysis of the 582del deletion region using the BLAST program revealed that it is not a highly conserved element but was found in the genomes of primate and ungulate species. " "582del deletion" is a repetition.
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